In the first wave of synthetic biology, genetic elements, combined into simple circuits, are used to control individual cellular functions. In the second wave of synthetic biology, the simple circuits, combined into complex circuits, form systems-level functions. However, efforts to construct complex circuits are often impeded by our limited knowledge of the optimal combination of individual circuits. For example, a fundamental question in most metabolic engineering projects is the optimal level of enzymes for maximizing the output. To address this point, combinatorial optimization approaches have been established, allowing automatic optimization without prior knowledge of the best combination of expression levels of individual genes. This review focuses on current combinatorial optimization methods and emerging technologies facilitating their applications. Our efforts to build complex synthetic biology circuits are impeded by limited knowledge of optimal combinations. In this review, the authors consider current combinatorial methods and look to emerging technologies. These circuits are now being combined to design regulatory circuits with complex performance, such as logical gates6. Often, multiple genes must be introduced into the host and expressed at appropriate levels to achieve the best possible output. Due to the enormous complexity of living cells, it is typically unknown at which level heterologous genes must be expressed, and to which level the expression of host-endogenous genes must be altered (if not deleted), to accomplish the goal7. Therefore, synthetic biologists aim to develop computational tools that can allow prediction of the performance of an assembly or an entire recombinant microorganism9. However, results from the computational analysis typically require validation through further wet-lab testing. This difficulty principally stems from the nonlinearity of biological systems10 and low-throughput characterization methods11. Furthermore, it is not always clear how to control noise12 and how to transfer the functionality of engineered elements between organisms7. Moreover, tweaking multiple factors can typically be critical to obtain an optimal output in a biological system13. Those may include overall structural state of chromatin and its domains14, the strength of transcriptional regulators controlling gene expression15, transcriptional terminators18, ribosome binding sites (RBS), biochemical properties of the protein(s) encoded by the recombinant genes21, the availability of cofactors for the correct functionality of enyzmes23, the genetic background of the host26, and the expression system itself 27.However, despite great developments of cutting-edge synthetic biology tools, engineering microorganisms for industrial scale production is still a challenging effort, even for well-known metabolic pathways28. The sequential flux maximization methodologies frequently utilize deletion of genes encoding competing pathways29. However, deletion of a gene can have broad physiological consequences that decrease cellular growth and productivity. For example, different levels of ArgR downregulation, achieved by CRISPR interference (CRISPRi), resulted in two times higher growth rates of Escherichia coli compared to deletion of ArgR30. However, increasing the production rate of a heterologous product in a recombinant microorganism is complicated and narrowing the research to debottlenecking strategies is too much of a simplification. For example, extensive work has been conducted to investigate the metabolism of the budding yeast Saccharomyces cerevisiae; nonetheless, there is still little progress in industrial scale production of high-value chemicals in this organism31. In one example, 244,000 synthetic DNA sequences were recently designed to uncover design principles of optimized translation in the well-known prokaryotic host E. coli32. Although impressive, this work provided little information about possible mechanisms underlying the improved translation capacity.To overcome these issues, two types of optimization strategies are available. The first one is “sequential optimization”, a classic method to optimize pathway performance33 and successful engineering of pathways is usually achieved only by trial-and-error7. Another approach to circumvent these barriers is to establish pathway “optimization” methods that do not require prior knowledge of the optimal expression levels of each individual gene involved in a multi-enzyme pathway. Several such methods have recently been developed, such as the functional optimization of gene clusters34, perturbation of the global transcription machinery35, genomic-scale mapping of fitness modifying genes36, multiplex automated genome engineering38, and “combinatorial optimization”. Jeschek et al. defined combinatorial optimization as “multivariate optimization” (in the context of metabolic engineering)7. The combinatorial optimization allows the rapid generation of a large number of diverse genetic constructs in short time7. Later on, to achieve high-level production of metabolites, microbial strains in a library that produce the highest level of a metabolite of interest need to be identified  or be used for single- or multi-locus integration into the microbial host genomes to generate combinatorial library (e.g. COMPASS39). Therefore, combinatorial optimization projects require tools and methods to assemble parts in genetic circuits, to change DNA sequences, and to integrate DNA pieces into the genome of an organism31. Here, we discuss two important synthetic biology tools: “advanced orthogonal regulators” and “advanced genome-editing tools”, as well as the recently established combinatorial optimization strategies.Combinatorial cloning methods aim to generate multigene constructs from libraries of standardized basic genetic elements such as regulators, gene coding sequences, and terminators using a series of one-pot assembly reactions8. Hence, auto-inducible (or self-induction) protein expression systems were established to pair growth and induction of recombinant proteins at desired time44. The system utilizes a cell density-based control module that allows tight regulation of the transcription of the recombinant gene; at low cell density, its expression is negligible while at high cell density expression is high. Such cell density-based control systems usually employ the quorum sensing (QS) mechanisms from Vibrio fischeri implemented in E. coli. However, these systems require the use of an extra plasmid for the production of proteins under control of the regulatory elements, which might be undesirable, particularly when multiple genes must be expressed. Another way of controlling the expression of regulators takes advantage of phage-derived anti-CRISPR proteins that allow fine-tuning the activity of dCas9-derived regulators at desired time points. The anti-CRISPR molecule inhibits the binding of Cas9 protein to DNA. This allows cells become resistant to further gene editing45. In another work, β-farnesene, pantothenate (a metabolic precursor of coenzyme A) was developed as a metabolic switch that effectively postpones metabolic burden until an optimal time for achieving maximal yield is reached46. Cultivation in media lacking pantothenate removes the growth advantage of low-producing mutants, resulting in improved production upon a scale-up to lab-scale bioreactors.Constitutive promoters are typically used to express heterologous genes in microbial cell factories, but this is often metabolically burdensome as the formation of the product competes with cell growth and proliferation47. More specifically, small RNAs can affect the chromatin structure allowing regulation of gene expression at the transcriptional level, or can affect mRNA stability post-transcriptionally or during translation. As a more sophisticated solution, orthogonal (and inducible) ATFs have been developed recently to control the timing of gene expression in various microorganisms48. To generate ATFs, the DNA binding domains (DBDs) of zinc finger proteins (ZFPs)49, transcription activator-like effectors (TALEs)15, and CRISPR/dCas915 scaffolds are used. Other DBDs, including LexA50, SrpR50, PhlF50, TarA50, Bm3R150, TetR50, auxin based degron51, FadRSa, CarH52, and plant TFs8 may be promising alternatives. However, unlike DBDs from TALE and CRISPR/dCas9 proteins, these DBDs cannot be easily modified to target any desired genetic sequence because they need to be paired with their own specific binding site. Nevertheless, ATFs have so far rarely been used in metabolic engineering projects due to their often large molecular size or low transcriptional activity. Surprisingly, plant-derived ATFs have recently been developed as strong regulators for S. cerevisiae (10-fold stronger than the yeast constitutive and strong TDH3 promoter)8. Expression of ATFs can be controlled by either exogenous chemical inducers 8 or by light of specific wave lengths53. However, hypersensitivity, toxic and pleiotropic effects limit the utilization of chemical inducers. An important task of future research is the identification of cost-effective inducers  that allow to control and modulate protein levels in response to a defined input signal in a fast-acting, tunable and robust manner. In this regard, light-based  systems have been developed that allow the expression of a gene of interest to an anticipated level by exposing the metabolite producing cells to short light pulses. Previous reviews have described diverse optogenetic control systems54. In Table 55. Such complexes have been largely employed as heterodimerization tools for small molecule switches. Unlike photoswitchable protein dimerizers  followed by homology-directed repair (HDR) is not high enough to support simultaneous generation of large numbers of integrations, even in a suitable host like 63. The combinatorial optimization method COMPASS allows the generation of a library of stable S. cerevisiae variants with thousands to millions of different members through only four cloning reactions followed by a one-step decoupled CRISPR-Cas9-mediated integration of the variants into the genome39. In a recent study, a method for the manipulation of the genome of mammalian cells was established through combining the CRISPR-Cas9 and the Cre-Lox systems64. In another effort, two optogenetic recombinases were developed for E. coli65. This approach uses split Cre and Flp  recombinases coupled with photodimers, where blue light brings the split protein together to form a functional recombinase. However, application of DSB-mediated genome editing is limited by the cellular cytotoxicity of HDR, unwanted DNA insertions and deletions, or unwanted additional DSB. To overcome these limitations, Barbieri et al. developed eMAGE (eukaryotic Multiplex Automated Genome Engineering) to precisely modify multiple sites of a genome without an involvement of DSBs. The method utilizes synthetic single-strand DNA (ssDNA) oligonucleotides targeting the lagging strand of the desired gene(s) in the replicating chromosome. Lambda (λ) phage Red Beta ssDNA annealing protein facilitates annealing of the ssDNA oligonucleotides to the lagging strand of the targeted DNA during DNA replication at the replication fork in S. cerevisiae66. Genome editing area of research can (i) shorten the gap between genome engineering and combinatorial library construction and (ii) the generation of diverse cell variants.A alternative is the CRISPR-Cas9 system (RNA-guided genome editing tool) which induce DSB and therefore allow higher efficiency in genome editingS. cerevisiae67.A multi-gene pathway can be combinatorially optimized at the DNA level by altering gene copy number, at the mRNA level by controlling transcriptional output and transcript stability, at the protein lLianevel by modifying translational capacity, protein post-translational modification, protein stability, and protein co-localization, at the metabolite level by dynamic metabolic flux control and at the chassis level by engineering microbial consortia. Lian et al. developed a combinatorial strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) for metabolic engineering purposes in S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. Yeast Golden Gate (yGG) is used to build transcription units (constitutive promoter upstream of a pathway gene) for VEGAS assembly. In the yGG reaction, each transcription unit is assigned a pair of VEGAS adapters that assemble up- and downstream of each transcription unit; VEGAS adaptor sequences subsequently provide terminal homology for overlap-directed assembly by homologous recombination “in yeast”. In contrast, inducible regulators allow conditional control of heterologous gene expression. Only a limited number of methods employing inducible and modular transcription factor-based controllers have been established so far, including e.g. ePathOptimize68 and COMPASS39. Combinatorial approaches typically rely on plasmid-derived systems to take advantage of the simplicity of manipulation at the plasmid level. However, genomic integration of a metabolic pathway leads to higher and more stable production of a target chemical than expression of genes from plasmids53, without the need for expensive selectable markers. In general, the chosen production host “determines” the regulatory elements , codon preferences, the way enzymes  are modified by posttranslational modification , and the biochemical mechanisms by which enzymes or metabolites are secreted out of the cell and into the growth medium (an important aspect of synthetic biology applications). Established combinatorial pathway optimization methods for metabolic engineering are summarized in Table S. cerevisiae, employs inducible plant-derived artificial transcription factors (ATFs) and utilizes three technical settings, (i) multi-locus integration of multiple genes into the yeast genome, when speed of strain development is prioritized, (ii) plasmid-based systems, when easy manipulation is favorable, and (iii) single genomic locus integration of multigene constructs, when predictable regulator behavior is required (as the genomic integration position affects the protein expression level)39.The majority of the research in the combinatorial optimization area has focused on transcriptional control mechanisms as an important control point for pathway gene expression. Currently available combinatorial approaches typically employ constitutive promoters, and therefore rely on growth-coupled biosynthesis. Versatile genetic assembly system (VEGAS) exploits the native capacity of 69. This system uses Cre recombinase and purified DNA encoding multiple loxPsym sites mixed in a tube. Wu et al.69 demonstrated two strategies using the “in vitro SCRaMbLE system” for pathway optimization: (i) The top-down method that consists of a purified DNA plasmid encoding multiple loxPsym sites. Addition of Cre recombinase to the reaction allows generating a library of SCRaMbLE DNA. (ii) The bottom-up system that employs an “acceptor vector” and a pool of “donor fragments” flanked by loxPsym sites. With the addition of Cre recombinase to the tube, donor fragments are randomly assembled into the acceptor vector. Therefore, a library of diverse plasmids is generated. The plasmid library can be subsequently transferred to a host strain for identification of genotype of each individual derivative. The in vitro SCRaMbLE system basically provides optimization in ways that the in vivo system cannot accomplish. However, application of combinatorial optimization strategies in in vitro production of proteins (cell-free protein synthesis (CFPS))70 or ex vivo 71 systems have not been studied yet. Such systems are typically used to produce toxic and/or large protein complexes, as there is no need to maintain cell viability. Combinatorial optimization strategies can be implemented to generate various combinations of genes and regulators in one cloning reaction cocktail. The in vivo and ex vivo systems require cellular lysate, transcription and translation factors72 to express different subunits of a large protein complex from the complex library of linear or plasmid DNA.Up to this point, we have highlighted the power of combinatorial optimization in order to facilitate expressing heterologous pathways in an in vivo system. The in vitro system relies on designing functional biological systems from scratch. Recently a straightforward in vitro method to smartly generate DNA library construction has been developed called “in vitro SCRaMbLE system” (an abbreviation for Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) enabling optimization of the biosynthetic pathway flux via rearranging relevant transcription units39 due to presence of repetitive sequences  in various ATFs.Linking the diversity within a combinatorial library at the DNA level to the production level of various members of combinatorial library can guide the debugging process to achieve the desired performance. However, the ability to capitalize on the combinatorial library diversity is limited by the number of individuals that can be tracked and assessed. To ascertain the diversity present in a combinatorial library, sequencing analysis is often used. While the identification of DNA diversity in small library can be achieved by Sanger sequencing, next-generation sequencing (NGS) has opened new ways to tackle complexity quality assessment of DNA diversity in a large library. Nevertheless, NGS is not suitable for identifying and characterizing ATFs implemented in COMPASS73. Microarray technology combined with molecular barcoding has been used to enable parallel tracking of genetically different individuals74. For example, trackable multiplex recombineering (TRMR) allows evaluation of thousands of definite genetic modifications in E. coli within a week. To do this, synthetic DNA cassettes with associated molecular barcodes were integrated into the E. coli genome, producing thousands of variants. Barcode sequences and microarrays were then used to compute population dynamics75. In another example, a pooled library of barcoded labelled mutants of Streptococcus pyogenes Cas9 (SpCas9) nuclease were easily tracked by high-throughput short-read sequencing76.Mapping the effects of protein mutation on its activity through employing high-throughput protein engineering methods or highly parallel mapping of genes to traits facilitated by multiplex DNA synthesis are now available and allow testing of a whole (or major fraction of) library population47. Deep sequencing, i.e. sequencing a genomic region multiple times, is an NGS approach that can be applied to track the genetic heterogeneity in a library of cell isolates. Höllerer et al. introduced a wildly applicable DNA-based phenotypic recording approach to generate huge datasets linking regulators to quantitative functional readouts of high precision, only relying on sequencing short tag DNA elements77. The technique implements a site-specific recombinase, a regulator that controls recombinase expression, and a DNA substrate modifiable by the recombinase. Both regulator sequence and substrate state can be determined in a single sequencing read, and the frequency of modified substrates amongst constructs harboring the same regulator presents the quantitative effect of regulator  on recombinase expression. Using next-generation sequencing, the quantitative expression effect of large library of regulators can be quantified in parallel. As a proof of principle, this approach was applied to record translation kinetics of more than 300,000 bacterial RBSs, collecting over 2.7 million sequence-function pairs in a single experiment. However, resolution of real genetic diversity in such a population can be an issue. Very recently, Askary et al. presented Zombie system for image-based readout of DNA barcodes75. In this system, phage RNA polymerases transcribe genomically integrated barcodes in fixed cells. The transcript RNA is then detected by fluorescent in situ hybridization. Single-nucleotide differences between barcodes are recognizable on the basis of the relative signal intensity of competing match and mismatch probes.We can extend the design principle of these methodologies to combinatorial libraries. Each DNA cassette encoding the individual fragment  can conceivably be labeled with a unique molecular barcode. Thereby, thousands of fragment combinations can be tracked by counting the frequency of the molecular barcodes. Moreover, sequencing short barcode fragments can significantly reduce the time and cost for sequencing regulators and downstream genes in individual isolates. However, to track the evolving genetic heterogeneity in a population of growing production cells, sequencing individual short barcode fragments is not sufficient and more advanced sequencing techniques such as “deep DNA sequencing” are requiredSelection of strains with the highest product yield from a library with huge genetic diversity is a serious bottleneck. Formation of colored products that make microbial colonies screenable is an option. However, most chemicals are not colorful and their detection requires other methods. In that case, direct quantification of product titer using low-throughput gas or liquid chromatography analysis is typically used despite being time-consuming. Methods based on spectroscopic enzymatic assay analytics are alternatives; however, they also have limited throughput. A more sophisticated solution is utilizing biosensor circuits to assay the target ligand at the single-cell level and translate their concentrations into more quantifiable signals. Biosensors are paired with high-throughput approaches, including flow cytometry and microfluidics, which allow isolation of variant cells with the phenotype of interest.79  implemented systematic engineering of multiple parameters to establish a general biosensor design in the yeast S. cerevisiae based on metasbolite binding transcriptional activators from the superfamily of LysR-type transcriptional regulators (LTTRs) of prokaryotes. They next used the biosensors to screen cells producing different level of naringenin or cis, cis-muconic acid. The designed biosensor output correlated with the production of metabolite. LTTR-based biosensors have already shown their utility to screen top producers: in one example they were used for screening 0.0000025% of the theoretical complexity of Narion (β-ionone and naringenin co-producer) library. Rogers et al. attempted to evaluate the tuning of four genetically encoded protein sensors that respond to acrylate, glucarate, erythromycin and naringenin on either low-copy or high-copy plasmids5. Higher number of the intracellular gene constructs ensured higher dynamic range as well as rapid response to the target ligand5. However, TF-based biosensors typically have relatively high background noise, and a lot of current research focuses on addressing this shortcoming. FRET biosensors encompass a pair of donor and acceptor fluorophores. A ligand-binding peptide is sandwiched between a pair of donor and acceptor fluorophores, and ligand binding is observed via the FRET change. A set of FRET biosensors, based on three pairs of donor/acceptor, carboxyfluorescein (FAM)/Boron dipyrromethene (BODIPY)85, Nitrobenzoxadiazole (NBD)/nonsteroidal dye (Nile red)86, and coumarine/NBD86, was developed for real time monitoring of acid sphingomyelinase at high sensitivity and with high spatial resolution. The FRET biosensor is selectively cleaved by sphingomyelinase that leads to significant increase in fluorescence of the fluorescein FRET donor85. FRET biosensors have high orthogonality, high temporal resolution, and relatively easy construction80. The RNA-based biosensors include RNA riboswitches and RNA Spinaches. In the case of RNA riboswitch-based biosensors, the regulatory domain of an mRNA selectively binds to a ligand resulting in a structural change to the response domain that regulates translation of its encoded protein. Abatemarco et al. developed RNA riboswitch-based biosensor to detect the production of tyrosine and streptavidin concentrations in S. cerevisiae library. The aptamer is co-encapsulated with a member of a yeast library followed by incubation to produce the molecule of interest and development of a fluorescence signal. The picoliter droplets flow through a microfluidic device allowing sorting based on fluorescence87. In comparison to TF based biosensors, their kinetics are faster because the RNA has already been transcribed and so offer faster responses to target metabolite. They also do not rely on protein-protein or protein-metabolite interactions. This allows for more targeted engineering of the aptamers (the ligand binding domain) and the expression platforms. The development of biosensors that allow in vivo evaluation of any desired product of metabolic engineering would be an absolute boon for the field. A step in this direction is the systematic evolution of ligands by exponential enrichment (SELEX) approach, which can be, at least in principle, used to generate artificial riboswitch-based biosensors (single-stranded RNA aptamer) for any target metabolites. Using SELEX, metabolite-responsive riboswitch-based biosensors are developed from a library of nucleic acids. The selected riboswitch-based biosensors are, subsequently, employed to detect and report the metabolite signal88.Only TF-79 Fig. , fluores 80 Fig. , and RNA 83 Fig.  based bi89. The small molecule–enzyme pair consists of uridine/cytidine kinase 2 and 2′-azidouridine, where 2′-azidouridine is only incorporated in cells expressing uridine/cytidine kinase 2. This pair can be used to purify and track RNA from specific cellular populations.Very recently, a high-resolution methodology for the cell-specific RNA labeling was established based on nucleoside–enzyme pair90. The directed (or adaptive laboratory) evolution improves the natural evolution process by (i) randomization of mutation or recombination and (ii) accelerated screening rate of positive strains90. It uses different databases, libraries of tools and conditions to generate the optimal production rate of a desired compound. Mathematical models and computational simulation could be applied as powerful tools to understand and predict the behavior of the biological systems.Metabolic engineering efforts aim to optimize the cellular processes for production of a compound of interest in host of choice -pinocembrin from screening of only 65 variants out of more than 23,000 possible combinations. Although this system was developed for E. coli, in principle it should be applicable for synthesis in other chassis. RBS Calculator allows the prediction of the translation initiation rate of internal start codons, particularly to minimize undesired expression in E. coli92.The multi-level omics data obtained from different databases for pre-characterized synthetic biology parts and modules are computationally integrated to establish mathematical models to support the early design steps of a system. The computational design  was recently implemented to evaluate various engineered strains for metabolites that contribute to acetol formation using metabolomics datasets. This data helped identify NADPH regeneration as the bottleneck for efficient acetol biosynthesis94. However, few laboratories work concurrently on both experimental and computational aspects of metabolic engineering, a fact that impedes the robust information exchange between these domains that is necessary for the highest productivity. RiboLogic tool was developed for designing riboswitches that are responsive to RNA inputs, as well as small molecule ligands3. RiboLogic algorithm was applied to design 286 switches that modulate MS2 (bacteriophage coat protein) binding of flavin mononucleotide, tryptophan, theophylline, and miR-208a, a 22-nt miRNA implicated in cardiac hypertrophythe.Several toolboxes are now available to integrate transcriptomics, proteomics, fluxomics and metabolomics data. Tn-Core allows automatic integration of Tn-Seq data in addition to RNA seq data to generate context specific models. This provides a “systems-level” view for metabolic engineering aims95. Höllerer et al., implemented next-generation sequencing to assess the quantitative expression effect of extremely large sets of RBSs77 . They expanded from these large-scale datasets using a novel deep learning approach that combines ensembling and uncertainty modelling to predict the function of untested RBSs with high accuracy. The data achieved from DNA-based phenotypic recording supports deep learning databases and, therefore, provides a major advance in our ability to predict quantitative function from genetic sequence.The use of selective marker genes as biosensors, links the host production performance with growth rate. Therefore, biosensors make possible to study directed evolution of the microorganism or the entire biological network .In particular, improvements in synthetic biology have made possible to study gene regulatory networks (GRNs) in a simplified setting amenable for exact experimental controls.In the past 30 years, a number of microbial processes have been developed for the production of such high-value chemicals including taxol64, bistable circuit of autoactivation104, or logic XOR and AND gates105. Combinatorial optimization coupled with biosensor techniques can facilitate generation of different possible GRNs, whose output can be measured and compared by placing a reporter gene downstream of the last regulator of the network and the output quantified by flow cytometry. This paves the way to understand regulatory relationships among genes and identifying key regulators and bottlenecks in GRNs.For example, researchers constructed mutual inhibition toggle between TetR and LacI inhibitors106. S. cerevisiae is a better production host than E. coli for production of amorphadiene, and β-farnesene due to less toxicity of pathway intermediates102, but producer populations were compromised by a high percentage of nonproducer mutants47. Hypothetical scenario to handle especially complex synthesis pathways is to divide them into pieces and design a different synthetic microbial host for each piece. The engineered microbes can then be grown together in consortia108. This can be substantially mitigated by utilizing combinatorial design principles to optimization multiple portions of the complex pathway (across multiple hosts) in parallel.There is no super host that is best for the production of all target moleculesSynthetic biology projects often need tunable expression of various combinations of genes to achieve an optimal output. Ideally, computational models will ultimately facilitate the forward-engineering of many parts to achieve desired function. However, until then the best alternative is to rapidly generate and accurately characterize as many new variants as possible. Therefore, smartly designed combinatorial optimization methods for the empirical balancing of metabolic pathway gene expression is of interest to a broad range of synthetic biologists. The workflow described in this review has the potential to become an enabling standard for researchers seeking to achieve optimal output while minimizing experimental effort in bioengineering projects. We expect that future developments in combinatorial optimization will still be limited by a lack of tools to screen the best producers from such a library. To continue advancing in our ability to engineer living organisms for high-level production of desired compounds using combinatorial optimization methods, we will require high-throughput screening techniques. Therefore, an interesting research direction would be a generalized biosensor construction framework for targeting diverse molecules. Ongoing advancements in developing riboswitch-based biosensors may become a larger part of the solution.75 or tracking of mutants of Cas9 nuclease library76. Such techniques however have yet to be applied for tracking of combinatorial library members. In fact, we can envision the use of the design principle of these methodologies to combinatorial libraries to identify the DNA elements combined in libraries of million circuits.In this review, we discussed how DNA barcoding methodologies have significantly improved in recent years, enabling the computation of population dynamicsCombinatorial optimization in synthetic biology encompasses a number of different techniques and disciplines that include molecular biology, analytical and protein chemistry and computational modelling. Progress in this field is emerging from a close interplay between bioinformaticians, chemists, and synthetic biologists.


Glial cells are an essential component of the nervous system of vertebrates and invertebrates. In the human brain, glia are as numerous as neurons, yet the importance of glia to nearly every aspect of nervous system development has only been expounded over the last several decades. Glia are now known to regulate neural specification, synaptogenesis, synapse function, and even broad circuit function. Given their ubiquity, it is not surprising that the contribution of glia to neuronal disease pathogenesis is a growing area of research. In this review, we will summarize the accumulated evidence of glial participation in several distinct phases of nervous system development and organization—neural specification, circuit wiring, and circuit function. Finally, we will highlight how these early developmental roles of glia contribute to nervous system dysfunction in neurodevelopmental and neurodegenerative disorders. Long past the days of being relegated to nervous system “glue,” today glia are acknowledged as essential players in nervous system development and function . Given tGlial cell populations can be subdivided based on lineage and function, most of which are conserved from invertebrates through humans , 2. DysfDrosophila) must divide for a stereotyped period of time. Their progeny, postmitotic neurons and glia, must then adopt distinct fates in proper numbers and stoichiometry before connecting with the appropriate partners to form functional circuits . These neural stem cells . This neuroepithelium is located at the surface of the developing optic lobe, where it generates the neurons of the medulla and lamina neuropils. The OPC proliferates and expands during the first and second larval instars before a proneural wave sweeps across the epithelium, resulting in a switch to asymmetric division for generation of neuroblasts  are not present during vertebrate neurogenesis in the embryonic central nervous system (CNS). In contrast, microglial populations migrate from the yolk sac into CNS during early embryonic development , and ared medium . Microgld medium , similartex glia . Furthertex glia . Finallyin vitro . In vivog retina . Here, kg retina .in vitro (in vitro and in vivo have since defined that complex extrinsic cues from astrocytes and oligodendrocyte precursor cells can bias adult NSCs toward neurogenic or gliogenic trajectories (–/– and microglia-specific knockout of MerTK/Axl) showed reduced hippocampal neurogenesis  . MacroceFragile X Mental Retardation 1 (FMR1) causes Fragile X syndrome, a disease associated with severe cognitive deficits and autism-like behaviors . PCD is essential for appropriate nervous system development. PCD eliminates excess, obsolete, or “damaged/incorrect” cells, and therefore serves as a means to adjust neuronal populations to refine circuit formation . DiffereOne of the earliest studies to define glia-derived signals that activate neuronal death was described in the chick embryonic retina. This pioneering work demonstrated that the secreted neurotrophic factor NGF (Nerve Growth Factor) is exclusively expressed by microglia during early embryogenesis and is required to induce cell death in the early retinal neuroepithelium . NGF canMicroglia play a similarly critical role in controlling cell death within the developing motor circuit. Motoneurons are born in excess in the vertebrate spinal cord . During Drosophila, neuronal cell bodies are surrounded by cortex glia that extend processes to envelop differentiated neurons. During development, correct morphology and physiology of cortex glia is required for the survival of new-born neurons  to upper cortical layers during early postnatal development , where they originate, to the most superficial cortex layer . This pattern of migration results in an inside-out organization, with early born cortical neurons in superficial layers and later-born neurons in deep layers. While excitatory neurons migrate radially, inhibitory interneurons populate the cortex by tangential migration from the ganglionic eminences to the cortex, slotting horizontally into the cortical layers . As mentelopment , showingSox10 , olfactory ensheathing cells do not develop and GnRH neurons fail to enter the forebrain, resulting in accumulation of GnRH neurons at the PNS-CNS boundary. Additionally, loss of olfactory ensheathing cells disrupts GnRH axon targeting, demonstrating a dual role for these glial cells in organization of the neural circuits underlying olfaction  neurons originate from the olfactory placode. GnRH neurons then migrate along the olfactory sensory neuron axons encompassed by the olfactory ensheathing cells into the developing forebrain . In the lfaction .Drosophila ventral nerve cord, analogous to the vertebrate spinal cord   . Vertebrnd Slit) . In vertnd Slit) . Correct in mice . Moreove in mice . In gene in mice .C. elegans has defined a glial cell population that instructs the initial assembly of the central nerve ring. The cephalic sheath (CEPsh) glia, which envelops the outer surface of the nerve ring once formed, extend processes to demarcate the nerve ring prior to arrival of any axonal projections. Once there, a bundle of fewer than 10 pioneer axons enter the nerve ring, followed by a stereotyped pattern of follower axons. By taking advantage of forward genetic screening approaches, it was shown that glial-derived Netrin and Semaphorin instruct pathfinding of pioneer and follower axons into the nascent nerve ring, respectively  exhibit over migration of neurons on radial glial processes and inverted layering of the mammalian cortex  regulation of synapse development, (2) selective pruning of supernumerary connections, and (3) continued synapse maintenance .C. elegans  . In thes elegans  and Drostrocytes  were simn region . In the n region . As thisn region , we willSynaptic pruning). Interestingly, depletion of microglia during early postnatal development, a period of rapid synaptogenesis, results in a significant reduction in spine density on Layer 2/3 pyramidal neurons in mouse (Il1rl1 (Interleukin 1 receptor like 1) from microglia, suppressed experience-dependent spine formation , which activate TGF-β receptors on motor axons to stimulate synaptic growth . In c growth . Similarc growth , and stic growth . For exac growth . Althougc growth . As 35% c growth , this stc growth  as a potDrosophila NMJ glia use Draper to phagocytose excess synaptic boutons on motor axons during NMJ development , which is highly dependent on glia  glia that support neuron receptive endings on thermosensing AFD neurons. Mutations in kcc-3 result in progressive retraction of neuron receptive endings , and Huntington’s disease suggest that downregulation of Kir4.1 in astrocytes may contribute to disease onset and/or progression  and increases animal survival  results in retraction of neuron receptive endings (pre-synaptic) that sense the environment  from peripheral glia results in abnormal nerve swelling and axonal defasciculation in Drosophila  within the blood are largely inaccessible to neurons due to blood-brain and blood-nerve barriers . Both cecomotion .in vitro within mouse organotypic spinal cord slice cultures, or in vivo within the optic nerve and corpus callosum, causes axonopathy prior to myelin damage  from Tdc2+ neurons in the ventral nerve cord activates Oct-TyrR on astrocytes, which in turn allows for calcium entry via Wtrw. Calcium influx drives secretion of an astrocyte-derived cue  that inhibits adenosine receptors on local dopaminergic neurons, which is necessary for proper larval chemotaxis and startle behavior. Thus, astrocyte-mediated neuromodulation occurs in vivo, is necessary for circuit function, and drives animal behavior  and globally (circuit-level). As this subject has been reviewed extensively, here we will direct readers to relevant literature , and insbehavior .in vivo within a behaviorally relevant circuit. Futility-induced passivity (“giving up”) is a strategy that allows animals to conserve energy following repeated failed attempts to achieve a given task , implicating gliosis as a putative target for therapeutic intervention (Analysis of human tissue revealed that microglial and astrocyte populations adopt activated, pro-inflammatory states locally and systemically in ALS . This swG93A , which cG93A . This chG93A . In the G93A , becauseG93A . MCT1 exG93A . A recenrvention .in vivo. By expanding the scope of our experiments to account for the complexity of these interactions, we have the potential to uncover novel therapeutic strategies to simultaneously inhibit glial inflammation and promote glial homeostatic functions to drive repair and recovery.To summarize, the nervous system contains a variety of glial cell types that collectively orchestrate neural development and ensure continued nervous system function. The field of glial biology has long focused on how individual glial subtypes influence neuronal health and longevity. Thus, we have achieved a great deal of success in delineating how disruptions to single glial cell populations contribute to pathology in various animal models of disease. As neurological disease states are often accompanied by dysfunction in multiple glial populations, it is unsurprising that therapeutic interventions that target individual glial and neuronal populations have been met with limited success. Furthermore, it is increasingly evident that in addition to glia-neuron interactions, dynamic glial-glial communication instructs development and function of the healthy and diseased nervous system. Generation of complementary genetic strategies that facilitate independent manipulation of multiple cell populations in vertebrates, while leveraging invertebrate models that are already amenable to complex genetics approaches, is necessary to understand how different glial subtypes function in concert with one another SA and VF conceived the topic. IL-B prepared the figures and together with VF and SA and wrote sections “Introduction” to “Glia in Neuronal Migration and Pathfinding of Axons and Dendrites.” SA wrote sections “Glial Regulation of Synapse Numbers” to “Concluding Remarks on Glia as Therapeutic Targets in Disease” with comments from IL-B and VF. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Germinal matrix-intraventricular haemorrhage (GMH-IVH), periventricular haemorrhagic infarction (PHI) and its complication, post-haemorrhagic ventricular dilatation (PHVD), are still common neonatal morbidities in preterm infants that are highly associated with adverse neurodevelopmental outcome. Typical cranial ultrasound (CUS) findings of GMH-IVH, PHI and PHVD, their anatomical substrates and underlying mechanisms are discussed in this paper. Furthermore, we propose a detailed descriptive classification of GMH-IVH and PHI that may improve quality of CUS reporting and prediction of outcome in infants suffering from GMH-IVH/PHI. The overall incidence ranges between 20 and 25% among very low birth weight (VLBW) infants.10 A venous origin of GMH-IVH has been demonstrated by postmortem studies12  on admission allows pre-existing antenatal brain injury to be identified. If antenatal GMH-IVH occurred well before birth, residual findings  may be subtle (see other relevant paper in this issue). PHI with an atypical time of onset  has been associated with the presence of thrombophilic disorders, especially factor V Leiden.17Foetal intracranial haemorrhage is not uncommon, with an estimated incidence of 0.5–0.9/1000 pregnancies. Antenatal GMH-IVH is the most common type of foetal intracranial haemorrhage.1Most instances of GMH-IVH/PHI are clinically silent and detected during routine CUS. Some infants manifest with subtle changes in the level of consciousness, limb movement, tone, and eye movement in the hours to days after the IVH. GMH-IVH and PHI can be accompanied by various degrees of cardiorespiratory instability and anaemia. With extensive haemorrhage, a catastrophic deterioration occurs with stupor, “decerebrate” posturing, generalized tonic seizures and hypotonia.40 and reflect the heterogeneity of this collective. Infants that suffer low-grade (i.e. grade I or II) GMH-IVH are clearly at much lower risk of developmental disabilities compared to infants with grade III GMH-IVH or PHI. Therefore, it has been customary to inform parents that the finding of an uncomplicated low-grade GMH-IVH has no relevant impact on later neurodevelopment and academic achievement. Recent data, though, suggest that this may not be entirely true.41 It has been shown that low-grade GMH-IVH is followed by microstructural impairment in periventricular and subcortical white matter.42 Size, number and location of these minor lesions might be of great importance in infants born at the lowest gestational ages, although robust data are lacking. Matrix injury even after an uncomplicated GMH-IVH results in a relevant loss of glial precursor cells, leading to impaired myelination and cortical development.44 GMH-IVH further triggers inflammation in adjacent white matter through activated microglia, passage of red blood cells and red blood cell degradation: the resulting perilesional tissue injury may be secondary to free radical release and the presence of free iron.48 The outcome of grade I and II GMH-IVH needs to be prospectively studied in relation to the exact description of size and location in different parts of the matrix protomap of telencephalic development. Similar relevant side effects of intraventricular blood products on the cerebellar external granular layer, after passage into the cerebellar subarachnoid spaces, are not discussed here.Infants with grade III GMH-IVH carry a significantly increased risk of neurodevelopmental disability, especially when GMH-IVH is complicated by post-haemorrhagic ventricular dilatation (PHVD) that needs surgical intervention. Cerebral palsy rates in infants with IVH III range between 7 and 63%50 GMH-IVH of all grades can be complicated by PHI, but the higher the grade of GMH-IVH, the more likely PHI is to occur.51 PHI is caused by venous obstruction induced by GMH-IVH. Venous congestion leads to ischaemia and to secondary haemorrhagic infarction. High intraventricular pressure due to a large haemorrhage may additionally affect flow through the subependymal veins, increasing infarct size.52 Cerebral palsy and severe cognitive impairment are common in infants who suffered from PHI.40 Prognosis is highly dependent on location and extent.56 Classifying PHI into venous subtypes helps to predict outcome and counsel parents in this difficult situation,55 and this should be expanded in relation to specific behavioural or cognitive sequelae. Mortality in infants with extensive PHI is high, especially when it occurs bilaterally. In many countries, redirection of care and end of life decisions are considered in infants with bilateral PHI. Although robust data on this are lacking, it is certain that redirection of care contributes significantly to reported mortality rates.58PHI complicates GMH-IVH in ~15% of cases.59 The risk of PHVD is higher following severe GMH-IVH (i.e. grade III GMH-IVH or PHI).60 The vast majority of instances of progressive PHVD (80%) follow IVH III, often in combination with a PHI. Most often it follows obstruction of liquor pathways around the cerebellum. While in most cases PHVD eventually resolves , around 35% of infants with progressive PHVD require surgical treatment, while 10% die.59 Despite decades of extensive research, treatment of PHVD remains challenging.61 Several options were investigated: lumbar or ventricular tapping, cerebrospinal fluid (CSF) drainage and fibrinolytic treatment, surgical insertion of an external drain, a subcutaneous reservoir and permanent ventriculo-peritoneal shunting. The key problem is to balance between the adverse effects of PHVD on the immature brain and the risk of complications of interventions .62 PHVD is strongly associated with neurodevelopmental impairment, particularly in infants with persistent PHVD that requires surgical intervention and if PHVD is combined with PHI.39 However, more recent studies revealed a better outcome than reported earlier.63 This might partly reflect the heterogeneity of management strategies among centres. A recent multicenter study showed that early treatment of PHVD, based on ventricular measurements, is associated with favourable neurodevelopmental outcomes, even when a permanent shunt is eventually needed.64The term PHVD refers to dilatation of the ventricles subsequent to GMH-IVH. Approximately 25% of infants with GMH-IVH develop progressive PHVD.65 In the most critical phase, CUS should be as “quick and gentle” as possible, in order to minimize stress for fragile neonates. As the incidence of GMH-IVH is closely related to gestational age at birth,66 it is reasonable to recommend the following schedule: in preterm infants with a gestational age below 28 weeks or 1000 g, serial CUS should be performed on days 1, 3, 7, 14, 21, 28, and then every other week until term-equivalent age. In stable preterm infants with a gestational age above 28 weeks, the frequency of serial CUS can be limited to days 1, 3, 7, 14,d 28, at 6 weeks and at term-equivalent age. Additional targeted examinations may be warranted in case of clinically or ultrasonographically suspected bleeding with uncertain findings.67 CUS examinations beyond the first week of life allow detection of PHVD as well as of uncommon occurrence of late-onset GMH-IVH.Preterm infants are often unstable during the first days of life, when GMH-IVH typically presents. CUS allows prompt diagnosis of GMH-IVH as well as assessment of the evolution.68 in 1978, based on severity assessed by computed tomography. Despite several ultrasound classifications subsequently published,70 the Papile classification has been widely used for decades by clinicians and researchers.71 It suggested that progression from a grade I to a grade IV haemorrhage represents a continuum, as follows: subependymal bleeding limited to germinal matrix (grade I), intraventricular haemorrhage extending into normal sized ventricles and typically filling <50% of the ventricular lumen (grade II), intraventricular haemorrhage extending into dilated ventricles (grade III), intraventricular haemorrhage with parenchymal extension (grade IV). However, the formerly called “grade IV” represents periventricular haemorrhagic venous infarction (PHI), rather than parenchymal extension by rupture into the parenchyma of the initial GMH-IVH—although this event very rarely does occur72  were lumped under grade I in Volpe classification, making the distinction between these two entities irrelevant.1 In light of the above-mentioned studies showing the potential detrimental effects of intraventricular blood on the developing brain, subependymal GMH and limited IVH were separated into two grades in the new classification system we propose in the present paper  in coronal planes occurring in the first postnatal days strongly suggests unilateral GMH. Of course, GMH may also occur bilaterally.72 Conversely, an echogenicity developing at the groove in the late neonatal period should suggest hyperechoic germinolysis rather than late GMH.77 Postmortem studies confirmed that the majority of GMHs develop in the caudo-thalamic region, although at postmortem GMH has been described also in occipital and temporal horns: these regions should be carefully examined during CUS.78 Neuroimaging studies have demonstrated that diagnostic accuracy of CUS for minor forms of GMH-IVH is suboptimal when compared to the MR-SWI sequence (magnetic resonance-susceptibility weighted imaging), which is considered the most sensitive technique for detection of subtle haemorrhage.79When bleeding is limited to the germinal matrix (GMH), the typical CUS finding is a subependymal hyperechoic globular thickening detected during the first week of life, which usually remains visible for a few weeks Fig. . As in s75 However, identification of a minimal amount of intraventricular blood  remains challenging. In this context, indirect signs can corroborate a diagnosis of IVH:80 hyperechoic ependymal changes appear from 2 to 4 weeks after IVH, while clots from supratentorial origin can be detected in the fourth ventricle and around the cerebellar surface by insonating through the mastoid fontanelle. Ventricular dilatation by large clot  should be distinguished from PHVD, which takes place after days or weeks due to distal obstruction of CSF circulation and perhaps later to impaired resorption.69 Ultrasonographic characteristics of clot changes over time should be always kept in mind in order to consider whether IVH could be of antenatal origin when subacute characteristics of the clot are observed soon after birth, or to suspect an IVH already in its earliest, hyperacute phase. Hyperechogenicity in the acute stage is due to fibrin formation at the end of the clotting cascade and is the main feature of the clot between 4 and 6 h and 3 days after the bleeding.81 In the earliest, hyperacute phase, fresh IVH may remain hypo- or isoechoic, and motion of particulate CSF can sometimes be visible within the ventricles (video particulate CSF with motion). In the subacute phase, after an initial retraction, clot is characterized by progressive hypoechoic change in the central portion and by hyperechoic margins. Intraventricular fibrin strands around the clot can be observed , typically at the stria terminalis. IVH can be “limited” or “extensive”: in practice “extensive” (grade III according to Papile and Volpe classification) is reserved for haemorrhage that leads to acute ventricle distension by clot and not by CSF, but this remains arbitrary, as measurement of clot volume is not possible with ultrasound. The presence of IVH is usually suggested by intraventricular hyperechoic clot located anteriorly to the foramina of Monro, above the caudo-thalamic groove or in the occipital horn. In the latter case, insonation through the posterior or even mastoid fontanelle allows better visualization of the clot.82 Plexus haemorrhage has been described associated to GMH-IVH in neuropathological studies of VLBW infants.83 However, discriminating plexus haemorrhage from GMH-IVH is difficult for the sonographer in the acute stage.Besides cerebellar haemorrhage, which is often detected in patients with GMH-IVH, an uncommon type of bleeding that can be associated with GMH-IVH is septal haemorrhage. The typical finding is the presence of a clot in the cavum septi pellucidi and/or in the cavum vergae Fig. . Septal 84 Postmortem and Doppler studies strongly suggest that this lesion is due to infarction following venous obstruction and congestion.87 It is also likely that arteriolar hypoperfusion secondary to venous obstruction contributes to parenchymal injury , also referred to as parenchymal haemorrhagic infarction or periventricular venous infarction or intraparenchymal lesion, can complicate each grade of GMH-IVH and seems to occur a few hours up to a few days after the initial bleeding.52  add useful information for prediction of outcome.93 Nevertheless, access to MRI is difficult in low-income countries, limited by obvious logistic obstacles in the acute or early subacute phase of PHI due to clinical instability of the infant. In this context, recognizing the venous subtype of PHI rather than labelling the lesion as an unspecified “PHI” (or “grade 4”) can help the clinician to predict neurological outcome, allowing the start of a targeted rehabilitation programme at an early stage, and may enrich the quality of counselling .59 For this reason, serial ultrasound scans are warranted following GMH-IVH at least until term-equivalent age  and anterior horn width (AHW), both obtained on a coronal plane passing through the foramina of Monro, are commonly used parameters. However, the absence of significant widening of the frontal horns may sometimes lead to under-estimation of PHVD severity, as neonates tend toward colpocephaly.101 Thalamo-occipital distance (TOD) is measured on the parasagittal plane and reflects the degree of dilatation of the trigone and the occipital horn of each lateral ventricle. Reproducibility of these ventricular measurements  was at least good in two ultrasound studies assessing both intra- and inter-observer reliability.100 VI and AHW are used in most European NICUs to define the threshold for PHVD treatment: although no international consensus exists on optimal timing, the majority of European Centres initiate treatment once the ventricular width has crossed the 97th percentile + 4 mm line on Levene’s graph.102 Common findings in severe PHVD include a rounded upper border of the frontal horns on coronal planes  and a rounded anterior profile of the third ventricle in the midsagittal plane, due to the markedly dilated infundibular and supraoptic recess. Moreover, a dilated fourth ventricle can be identified in the midsagittal plane in case of tetraventricular or even communicating hydrocephalus. Besides these basics, the routine use of additional acoustic windows can provide useful information to understand the type of PHVD the patient has developed: insonation above the ear, can show a markedly dilated aqueduct in tetraventricular or communicating hydrocephalus, as well as an obstructed aqueduct in triventricular hydrocephalus.75 By gently tilting and rotating the probe on a pseudo-axial plane, the sonographer should aim to depict, in the same image, third and fourth ventricle connected by the aqueduct  may lead to more precise prediction of outcome and open new horizons in the understanding of pathophysiology of brain injury and complications related to GMH-IVH.Video


The transcriptional co-regulators YAP and TAZ pair primarily with the TEAD family of transcription factors to elicit a gene expression signature that plays a prominent role in cancer development, progression and metastasis. YAP and TAZ endow cells with various oncogenic traits such that they sustain proliferation, inhibit apoptosis, maintain stemness, respond to mechanical stimuli, engineer metabolism, promote angiogenesis, suppress immune response and develop resistance to therapies. Therefore, inhibiting YAP/TAZ- TEAD is an attractive and viable option for novel cancer therapy. It is exciting to know that many drugs already in the clinic restrict YAP/TAZ activities and several novel YAP/TAZ inhibitors are currently under development. We have classified YAP/TAZ-inhibiting drugs into three groups. Group I drugs act on the upstream regulators that are stimulators of YAP/TAZ activities. Many of the Group I drugs have the potential to be repurposed as YAP/TAZ indirect inhibitors to treat various solid cancers. Group II modalities act directly on YAP/TAZ or TEADs and disrupt their interaction; targeting TEADs has emerged as a novel option to inhibit YAP/TAZ, as TEADs are major mediators of their oncogenic programs. TEADs can also be leveraged on using small molecules to activate YAP/TAZ-dependent gene expression for use in regenerative medicine. Group III drugs focus on targeting one of the oncogenic downstream YAP/TAZ transcriptional target genes. With the right strategy and impetus, it is not far-fetched to expect a repurposed group I drug or a novel group II drug to combat YAP and TAZ in cancers in the near future. The transcriptional co-regulators YAP (Yes-associated protein) and TAZ  are key players that mediate various oncogenic processes and targeting their activities has emerged as an attractive option for potential cancer therapy. YAP, as the name suggests, was initially identified as a protein that associates with Yes, a src family kinase (SFK) YAP and TAZ do not have a DNA-binding domain and they need to associate with a transcription factor in order to access DNA. It has now emerged that YAP/TAZ use predominantly the TEAD (TEA domain) family of transcription factors Drosophila but the pathway components are evolutionarily conserved. It is now known that the primary function of the Hippo pathway is to suppress the activity of Yorkie - the Drosophila homolog of YAP YAP/TAZ are now considered as effectors of a physiologically and pathologically important signaling pathway - popularly called the Hippo pathway YAP and TAZ were first shown to transform mammary epithelial cells Over a decade of research has revealed that YAP/TAZ integrates the inputs of various oncogenic signaling pathways, such as EGFR, TGFβ, Wnt, PI3K, GPCR and KRAS. Through expression of the ligand AREG, YAP was first shown to communicate with the EGFR pathway YAP and TAZ regulate the expression of crucial transcription factors like Sox2, Nanog and Oct4 and are able to maintain pluripotency or stemness in human embryonic stem cells (ESCs) and in induced pluripotent stem (iPS) cells A TAZ fusion gene (TAZ-CAMTA1) alone, in the absence of any other chromosomal alteration or mutation, is sufficient to drive epithelioid hemangioendothelioma (EHE), a vascular sarcoma There are more than fifty drugs that have been shown to inhibit YAP/TAZ activity Group I drugs target the upstream proteins Figure , inhibitDrugs targeting the EGFR, GPCR, Integrin, VEGFR and adenylyl cyclase families as well as those targeting receptors like the γ-secretase complex and Agrin are shown to inhibit YAP/TAZ activity YAP/TAZ exploits the transformative abilities of the ErbB receptors (EGFR family) to drive cell proliferation. By transcribing ErbB ligands, such as AREG Signaling from G-protein coupled receptors (GPCRs), transduced by the associated Gα subunit or by the Gβγ subunits, modulates YAP/TAZ activities Integrin signaling negatively regulates the Hippo pathway complexes to drive YAP/TAZ activity Among the kinase inhibitors tested in a biosensor screen for LATS activity, the VEGFR inhibitors are shown to potently activate LATS and thereby inhibit YAP and TAZ activity Enhancing cyclic AMP (cAMP) levels using the adenylyl cyclase activator forskolin activates the LATS kinases through Protein kinase A (PKA) and Rho Notch and YAP/TAZ signaling are also closely linked, inhibiting notch activity by targeting the γ-secretase complex, either using DAPT or dibenzazepine has been shown to decrease YAP/TAZ expression levels in mouse livers and also reduce YAP activation and YAP-induced dysplasia in the intestine Integrin signaling activates focal adhesion kinase (FAK), SFK and integrin- linked kinase (ILK). Growth factor and GPCR signaling occurs through mitogen-activated protein kinase (MAPK) and phosphoinositide 3-OH kinase (PI3K) signaling. There is also significant crosstalk in the signaling from these membrane receptors. Given the availability of potent small molecule drugs targeting the downstream kinases, they are leveraged on to inhibit YAP or TAZ activities.Members of downstream integrin signaling pathway including FAK, its counterpart PYK2, and ILK have emerged as negative regulators of the core Hippo pathway and thus activate YAP/TAZ. Membrane receptors, such as ErbB and GPCRs are unable to activate YAP upon genetic deletion of ILK. Therefore, pharmacological inhibition of ILK using a specific ILK inhibitor, QLT0267 potently inhibits YAP-dependent tumor growth in xenograft models The SFK member Src prevents the activation of LATS MEK (MAP kinase kinase) and YAP interact with each other and maintain transformed phenotypes in liver cancer cells PI3K inhibitors Wortmannin/LY294002 as well as the drug BX795, an inhibitor of its effector 3'-phosphoinositide-dependent kinase-1 (PDK1) prevents nuclear entry of YAP YAP/TAZ inhibition is an additional unexpected activity possessed by the few kinase inhibitors mentioned above. However, apart from YAP/TAZ inhibition, all other signaling events initiated by the target kinase are also shut down due to inhibitor treatment. If these signaling events are critical for cellular homeostasis, then, toxic side effects will outweigh clinical benefits and this cannot be uncoupled from YAP/TAZ inhibition. Therefore, kinase inhibitors that failed in the trials due to unacceptable toxcity or poor pharmacokinetics may not be repurposed as YAP/TAZ inhibitors in the clinic. Focus should be on the kinase inhibitors that are already in the clinic like EGFR, VEGFR, MEK, PI3K or mTOR inhibitors but efficacy needs to be proven in order to repurpose them as YAP/TAZ inhibitors. The kinase targeted by the inhibitor must activate YAP/TAZ in tumors, for the treatment to be efficacious and this restricts the use of kinase inhibitors to selective tumor types. Intriguingly, YAP/TAZ activation has emerged as a prominent survival strategy adapted by cancers that cause drug resistance to EGFR and its downstream MEK/MAPK inhibitors The mevalonate pathway is essential for the biosynthesis of isoprenoids, cholesterol and steroid hormones. Statins as well as other mevalonate pathway inhibitors like zoledronic acid and GGTI-298 that target farnesyl pyrophosphate synthase and geranylgeranyltransferase, respectively are identified as drugs that restrict the nuclear entry of YAP and TAZ Actin polymerization promotes YAP/TAZ nuclear localization and therefore, polymerization inhibitors like latrunculin A YAP/TAZ inhibitory phosphorylation is dynamic and the protein phosphatases PP1 and PP2A are shown to associate with YAP/TAZ and aid in their dephosphorylation and activation. Inhibiting these phosphatases using okadaic acid or calyculin A increases YAP/TAZ phosphorylation and shifts YAP/TAZ to the cytoplasm Cellular energy stress is closely linked with attenuation of YAP/TAZ activities Histone deacetylases (HDACs) are uniquely positioned to alter the transcription of target genes. Interestingly, HDAC inhibitors panobinostat, quisinostat, dacinostat, vorinostat and Trichostatin A transcriptionally repress the expression of YAP but not TAZ, and thereby reduce YAP-addicted tumorigenicity Many group I drugs can potentially be repurposed to treat YAP/TAZ- driven cancers Modalities that target either the TEAD family of transcription factors or YAP/TAZ are classified under this group Figure . The majGroup I drugs target the upstream YAP/TAZ-activating proteins like the EGFR, GPCR, Src, or Integrins. As there are so many upstream YAP/TAZ activators, group I drugs are vulnerable to oncogenic bypass where inhibition of one group I YAP/TAZ activator leads to selection of cancer cells that activate YAP/TAZ via another group I activator. Strategically, Group II drugs may address this issue by directly targeting YAP/TAZ or TEAD, the converging points for various pathways and also the effectors for oncogenic transcription. However, Group II targeting modalities are still at the exploratory stage and it remains to be seen whether it is feasible to develop a Group II modality that works in clinic. We also need to be mindful of the possible associated toxicities due to YAP/TAZ-TEAD inhibition Most of the reported Group II modalities are disruptors; they target YAP/TAZ or TEAD and prevent their binary interaction. However, in addition to disruptors, in the future, we predict the emergence two other classes of group II compounds that would act as TEAD stabilizers and destabilizers/degraders Figure .A small molecule benzoporphyrin drug named Verteporfin (VP) was shown to have the ability to bind to YAP and disrupt the YAP-TEAD interaction YAP and TAZ bind on the TEADs' surface; Inventiva Pharma has identified several compounds with benzisothiazole-dioxide scaffold that bind to the TEADs' surface and disrupt the YAP/TAZ-TEAD interaction. These compounds are currently in the lead optimization stage and have the potential to treat malignant pleural mesothelioma as well as lung and breast cancers that are driven by YAP/TAZ in vitro but they have poor cell-penetrating abilities YAP cyclic peptide (peptide 17) and cystine-dense peptide (TB1G1) are also disruptors of YAP/TAZ-TEAD interaction We identified a novel druggable pocket in the center of the TEADs' YAP/TAZ- binding domain Palmitate, by occupying the PBP, allosterically modulates YAP's interaction with TEAD Being predominantly unstructured, YAP and TAZ are difficult to target directly. However, TEADs offer two attractive ways for targeting, one is to directly block the YAP/TAZ-binding pocket on the TEADs' surface with small molecules or peptides, whilst the other is to leverage on the PBP and allosterically disrupt YAP/TAZ interaction or inhibit TEADs Figure . HoweverThe PBP could also be leveraged to allosterically enhance YAP/TAZ-TEAD stability or interaction. This prediction is subject to the identification of small molecules that functionally mimic the ligand palmitate Figure . Compounin situ unfolding and degraders reduce TEAD levels through proteasomal degradation. Reducing the levels of their interacting partners deprives YAP/TAZ of their ability to activate transcription.We identified a few chemical scaffolds that have the ability to occupy the PBP and destabilize TEAD (unpublished results). Addition of these compounds unfolds the TEADs' YAP/TAZ-binding domain and we call these compounds destabilizers Figure . DegradeAny TEAD-binding compounds  can only access unbound TEADs, as binding of YAP and TAZ blocks both the surface and the palmitate-binding pockets Figure . After aYAP/TAZ-mediated tumor development is due to the collective action of the repertoire of proteins that are expressed under their influence. However, some proteins are able to drive oncogenesis much better than others and they vary depending on the solid tumor and context. Therefore, drugs against these downstream YAP/TAZ targets including metabolic enzymes, kinases, ligands and proteins, such as BCL-xL, FOXM1 and TG2 are also used to combat YAP/TAZ-mediated oncogenicity  is shown to impart stemness and tumorigenic ability; inhibition of ALDH1A1 using A37 reverses this transformation In hepatocellular carcinoma, Axl kinase has been shown to be crucial for mediating several YAP-driven oncogenic functions like cell proliferation and invasion In a mouse model of prostate adenocarcinoma, the YAP-TEAD complex promotes the expression of the chemokine ligand CXCL5 that facilitates myeloid-derived suppressor cells (MDSC) infiltration and adenocarcinoma progression. Administering CXCL5 neutralizing antibody, or blocking CXCL5 receptor using the inhibitor SB255002, inhibits MDSC migration and tumor burden YAP mediates drug resistance to RAF- and MEK-targeted therapies in BRAF V600E cells, in part through the expression of the anti-apoptotic protein BCL- xL. BCL-xL inhibition using navitoclax sensitizes these cells to targeted therapies YAP-mediated proliferation through its target gene FOXM1 could be prevented in sarcoma cell lines and mouse models through the administration of thiostrepton that reduces FOXM1 levels Transglutaminase 2 (TG2) - the multifunctional transamidase is a YAP/TAZ target gene that is important for cancer stem cell survival and for maintaining integrin expression. TG2 inhibition using NC9 dramatically reduces tumorigenicity We are aware that many of these target proteins also act upstream and stimulate YAP/TAZ by forming a positive feedback but we would nevertheless consider them in this group and not as group I as their expression is influenced by the TEAD-binding motif and YAP/TAZ.Although attractive, toxicity issues and the identification of responsive patient population could be challenges in the successful implementation of the YAP/TAZ inhibitors in the clinic. YAP/TAZ inhibition might elicit toxicity Several drugs that act as YAP/TAZ inhibitors target multiple signaling pathways. Targeting multiple pathways could be a boon or a bane. Drug resistance is minimized in a multi-targeted approach as potential bypass mechanisms are also targeted. However, toxicity becomes an issue when the drug targets multiple important signaling pathways. For instance, raising cAMP through the use of PDE inhibitors activates a multitude of proteins like PKA, EPACs, ion channels and small GTPases. Similarly, GPCR modulators influence multiple pathways through signaling via G proteins, arrestins or GPCR kinases. To reduce toxic side effects, there are options available like selective targeting or biased signaling. Instead of hitting all the PDEs, the PDE enzyme that is the most potent activator of YAP/TAZ should be selectively targeted. Nonspecific PDE inhibitors cause more severe side effects than sub-type selective PDE inhibitors Another major challenge is the identification of patients responding to a YAP/TAZ inhibitor. YAP/TAZ expression is low in normal tissues and their levels are significantly elevated in cancers. Is YAP or TAZ positivity in tumors sufficient criteria to administer a YAP/TAZ inhibitor? YAP and TAZ might not be transcriptionally active or drivers in all tumors. Further, they could be expressing target genes that negatively regulate their activity As YAP and TAZ contribute to the acquisition of many hallmarks of cancer traits, targeting them is predicted to be more relevant for the management of several cancer types. It is still early to expect a newly developed drug against YAP/TAZ but it is nevertheless disconcerting to see that there are hardly any clinical trials that evaluate if known drugs could be repurposed as YAP/TAZ- inhibitors. Group I drugs are well suited to repurpose


The results from these studies have identified amultitude of shear stress-regulated molecular networks in the endothelium that areimplicated in health and disease. This review outlines the significance of scientificfindings generated in collaboration with Dr. Shu Chien.Lining the luminal surface of the vasculature, endothelial cells (ECs) are in directcontact with and differentially respond to hemodynamic forces depending on their anatomiclocation. Pulsatile shear stress (PS) is defined by laminar flow and is predominantlylocated in straight vascular regions, while disturbed or oscillatory shear stress (OS) islocalized to branch points and bifurcations. Such flow patterns have become a centralfocus of vascular diseases, such as atherosclerosis, because the focal distribution ofendothelial dysfunction corresponds to regions exposed to OS, whereas endothelialhomeostasis is maintained in regions defined by PS. Deciphering the mechanotransductionevents that occur in ECs in response to differential flow patterns has required theinnovation of multidisciplinary approaches in both  The arterial cardiovascular system is made up of a luminal endothelial layer that isessential to vascular health by responding to and relaying mechanical, paracrine, andendocrine stimulations to circulating macrophages and underlying smooth muscle cells(SMCs).in vitro using a parallel plate flow system[2 for PS and0.5 ± 4 dynes/cm2 for OS), similar to those occurring under physiologicalconditions [in vitrostudies, in vivo animal experiments were conducted by comparing thethoracic aorta to the aortic arch to investigate the effects of PS and athero-protected flowvs OS and athero-prone flow, respectively [Endothelial cells are mechanosensors that respond to physical forces caused by local bloodflow patterns. Pulsatile shear stress (PS) occurs at straight regions of a vessel and ischaracteristically unidirectional. PS promotes EC homeostasis and vascular health. Incontrast, oscillatory shear stress (OS) is associated with disturbed flow patterns thatoccur at vessel curvatures or bifurcations. OS induces a local inflammatory environment,promoting site-specific initiation and progression of atherosclerosis. These local shearstress patterns can be modeled w system. In thisnditions . Using tectively .AdditioIn vitro and in vivo systems were used to investigate theprocess by which shear stress spatial-temporally regulates mechanosensors, signalingmolecules, and gene regulation to influence EC phenotypes.A significant pathway is the PS-induced KLF2 and KLF4 that are important transcriptionfactors in PS induced EC function in part by transcriptionally activating endothelial nitricoxide synthase (eNOS).A hallmark of atherosclerosis is the accumulation of foam cells in the vascular wall. Underthe conditions of OS, ECs have an augmented expression of monocytic chemoattractant protein1 (MCP1).OS also affects EC lipid metabolism. Sterol regulatory element-binding proteins (SREBP) 1and 2, which are key transcription factors that transactivate genes related to cholesterolbiosynthesis and uptake, are upregulated under OS.in vivo,Yes-associated protein (YAP) and its related coactivator, PDZ-binding motif (TAZ), are keymediators of organ size and have been implicated in OS-induced EC proliferation.Taken together, these studies established that the mechanotransduction induced by flowpatterns plays a major role in determining the atherogenic or atheroprotective ECphenotypes.in vivo applications and translational science. These fundamental issueswere addressed by multidisciplinary approaches that included a close collaboration betweenexperts in experimental science, medicine, engineering, and bioinformatics. These visionaryperspectives were later reflected in an elegant and highly cited review written forbioengineers.EC responses to mechanical stimulation are governed by multiple signaling events that havenot been attributed to a single cellular receptor. Early investigations of these events werelimited to the available genetic and molecular biology methodologies, which did not allowassessments of cellular functions at a broad level. A global view became possible with theuse of new integrative techniques such as next-generation sequencing (NGS). However, thesenew technologies posed difficulties in their application to interpret the large datasets inAmong the molecular players in EC health, AMPK has been studied as a key regulator of EChomeostasis. The application of bioinformatic approaches has identified novel AMPK targetsat the proteomic level and previously unknown functions that advanced our understanding ofendothelial function in atherosclerosis.In addition to identifying new signaling pathways at the proteomic level, bioinformaticsplatforms have been developed to compile information from publicly available large datasetsto facilitate the resolution of mechanisms that orchestrate non-coding RNAs, chromatinremodeling, and gene expression. As microRNAs (miRNAs) began to emerge as novel regulatorsin mammalian cells, NGS and bioinformatics approaches were leveraged to interrogate the roleplayed by miRNAs in EC mechanobiology. Integrating approaches developed throughbioengineering, RNA biology, and cardiovascular physiology, a number of mechanosensitivemiRNAs were identified as crucial players in EC biology.The growth of knowledge has led to the concepts that flow not only regulates the EC miRNAtranscriptome, but also modulates the secretion of EC miRNAs and their serving as“messengers” to other vascular cells, such as the vascular smooth muscles cells.lncRNAs have become known as crucial regulators of EC functions. Integration oftranscriptome and chromatin conformation capture profiling  has led to the identification of lncRNAs as important epigenetic modulators of ECresponses to shear stress. For example, the PS-induced enhancer-associated lncRNA thatenhances eNOS expression (LEENE or LINC00520) increases eNOS,Studies on PS-induced epigenetic regulation in ECs have identified KLF4 as a keytranscription factor in shear-regulated EC homeostasis. NGS profiling using RNA-seq,H3K27ac, and H3K4me chromatin immunoprecipitation sequencing (ChIP-seq), and assay fortransposase-accessible chromatin using sequencing (ATAC-seq) was conducted following shearstress stimulation to explore KLF4-mediated chromatin remodeling and the resulting geneexpression. To accomplish this novel endeavor, the acquisition and analysis of these datarequired cross-disciplinary coordination that includes bioinformatics, molecular biology,and bioengineering. After extensive analyses, it became clear that PS- and OS-inducedH3K27ac enrichment was associated with adjacent gene expression in the ECs of 18 novelPS-upregulated genes, the most significant being inositol 1,4,5-trisphosphate receptor type3 (ITPR3). Consistent with these findings, KLF4 ATAC-seq resolved a specific locus in thepromoter region of the ITPR3 gene that was essential for chromatin accessibility of KLF4binding and transcriptional activation. Deletion of this KLF4 binding locus in ECs usingclustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9(Cas9) blunted eNOS expression and diminished NO bioavailability .91 TakenCollectively, the discoveries emphasized in this review encompassed research conducted incollaboration with Dr. Shu Chien and how it integrates into the field of vascular biology.Combined with the work of others, these findings contributed to a paradigm shift in thefield of mechanotransduction by resolving mechanisms demonstrating the active role ECs playin the pathogenesis of vascular-related disorders, such as atherosclerosis. These seminaladvancements were enabled in part through the application of innovative approaches thatamalgamate disciplinary boundaries from cellular and molecular levels to translationalvascular medicine that pave the way for new vascular biology studies in the wholeorganism.Responding to local mechanical cues that synergize with stimulations originating throughoutthe organism, endothelial cells orchestrate cellular responses in vascular and subvascularregions that are implicated in a number of diseases such as pulmonary hypertension,Alzheimer's disease, vascular dementia, and pancreatic cancer.


RANKL and RANK are expressed in different cell types and tissues throughout the body. They were originally described for their essential roles in bone remodeling and the immune system but have subsequently been shown to provide essential signals from regulating mammary gland homeostasis during pregnancy to modulating tumorigenesis. The success of RANKL/RANK research serves as a paragon for translational research from the laboratory to the bedside. The case in point has been the development of Denosumab, a RANKL-blocking monoclonal antibody which has already helped millions of patients suffering from post-menopausal osteoporosis and skeletal related events in cancer. Here we will provide an overview of the pathway from its origins to its clinical relevance in disease, with a special focus on emerging evidence demonstrating the therapeutic value of targeting the RANKL/RANK/OPG axis not only in breast cancer but also as an addition to the cancer immunotherapy arsenal. In 1889, the “seed and soil” theory was first proposed by Stephen Paget for tumor metastases to distant organs . Bone isThe receptor activator of the nuclear factor kappa-B ligand , together with its receptor RANK (TNFRSF11A) , the decBone provides strength and structure, protects vital organs, stores minerals such as calcium, and is essential in the production of hematopoietic cells. Bone homeostasis is maintained by the balance between mainly two types of cells: osteoblasts  which build bone; and osteoclasts (derived from bone marrow hematopoietic precursor cells) which resorb bone  Figure . OsteoblRANK  , 12, 13 + vesicles bind membrane-bound RANKL on the osteoblasts and thereby promote bone formation by triggering RANKL reverse signaling via activation of Runt-related transcription factor 2 (Runx2). Targeting RANKL reverse signaling represents a novel strategy to avoid the reduced bone production associated with inhibition of osteoclastogenesis  acts as a decoy receptor for RANKL and is induced by estrogen, IL-4 or transforming growth factor beta (TGF-β) . OPG comogenesis .As RANKL is an important regulator of bone loss in bone metastases (associated with cancers such as multiple myeloma) and in postmenopausal osteoporosis, a specific, fully human IgG2 monoclonal RANKL antibody (mAb) has been developed, which neutralizes the activity of RANKL, which has been designated as Denosumab. The efficacy of Denosumab has been confirmed in multiple clinical trials, and Denosumab therapy is now approved and widely used for the treatment of various bone-associated diseases –45.Apart from bone homeostasis, the RANKL/RANK/OPG axis is also involved in various physiological immune processes. RANK was originally discovered on dendritic cells (DCs), and RANKL mediates the survival of DCs . The intrankl and rank-deficient mice revealed a complete absence of peripheral lymph nodes but intact spleen and Peyer's plaque structures  maturation involved in central immune tolerance  numbers  and RANKL , 66. In ignaling .Breast cancer is the most prevalent female malignancy . Studiesrankl knockout mice, our group was the first to report that during pregnancy, RANKL deficiency results in a total block in the development of lobuloalveolar milk-secreting structures   breast tumor mouse model  could potentially be used for the prophylactic treatment of breast cancer in carriers . Indeed  ongoing . Another ongoing , found n ongoing .BRCA1 mutation-driven mammary cancer tumorigenesis, it has also been reported that this pathway can induce epithelial-mesenchymal transition (EMT) in breast cancer cells, as well as in prostate and endometrial cancers  that can affect tumor growth, tumor cell dissemination, and metastasis . RANKL eBRCA1 mutation carriers, revealing a novel pathway whereby ERRα in primary breast cancer could promote early dissemination of cancer cells to bone (p = 0.01) and patients within the highest quartile of RANKL had a significantly increased risk of developing bone metastases compared to those in the lowest  , an important factor of cancer cell invasiveness, promotes breast cancer cell dissemination from primary mammary tumors to the bone . Intrigu to bone . Moreove = 0.03) . This st = 0.03) .In light of the different roles of the RANKL/RANK pathway in bone metabolism and immune system functions, therapy targeting this axis may not only control primary tumor development such as in the case of breast cancer and reduce bone metastasis which has been demonstrated in clinical trials , 153 butIn randomized clinical trials, Denosumab has shown rapid effectiveness by directly impairing osteoclast activity and inducing osteoclast apoptosis . Moreoven = 702) treatment with Denosumab showed a significant improvement in overall survival (n = 1718) also demonstrated the effectivity of Denosumab to reduce the occurrence of primary SRE events; moreover, the use of Denosumab significantly improved progression-free survival (PFS)  in patients with solid tumors  and bone metastases, the results showed that Denosumab was similar to ZA in preventing or delaying the onset of primary SREs , 157. Hosurvival . In thessurvival . The resal (PFS) . Whetherp < 0.0001) (p = 0.026). These data suggest that adjuvant Denosumab can significantly improve the DFS rate of HR+ postmenopausal breast cancer patients  , 158. A patients . Howeverpatients . These ipatients , 153. Itpatients .The field of cancer immunotherapy has paved the way for a new paradigm to combat cancer, by coaxing the body's own immune system to seek out, specifically target, and destroy cancer cells. Among the various approaches, immune-checkpoint inhibitors that target CTLA-4 as well as the PD1–PDL1 interaction, enabling an enhanced T cell-dependent anti-tumor response, have revolutionized the treatment of advanced melanoma and other cancers. However, using anti-PD1 monotherapy or the combination of anti-PD1 and anti-CTLA4 is still limited to a minority of cancer patients, and a vast majority of patients do not derive clinical benefit due to primary resistance . Thus, nBRCA1 mutations. Recent data have recently hinted at the exciting possibility of further repurposing Denosumab from an anti-resorptive agent in bone diseases to cancer immunotherapy in combination with checkpoint inhibition  immune tolerance to activate tumor-specific T cells after they have left the thymus. However, much less is known about how central  tolerance inhibits anti-tumor immunity. As mentioned above, the RANK–RANKL axis is required for the development of the AIRE-expressing medullary thymic epithelial cells (mTECs) of the thymus, involved in the “self” education of developing T cells . It has  T cells . As mTECBRCA1-mutated associated breast cancer. Since thousands of women have received the RANKL blocking antibody Denosumab to treat osteoporosis thus highlighting its safety profile, we believe that targeting the RANKL/RANK pathway with Denosumab is indeed a feasible strategy for the prevention of breast cancer in BRCA1-mutation carriers and may also be effective for other women at high risk for developing breast cancer. Importantly, a multinational phase III clinical trial using Denosumab to prevent the development of breast cancer in BRCA1-mutation carriers is currently being initiated. Recently, anecdotal evidence from case studies has raised the fascinating prospect of using RANKL inhibition as a novel cancer immunotherapy. Further research is however needed to reveal the underlying mechanisms of how RANKL blockage enhances anti-tumor responses of the immune system and whether combinatorial treatment with known checkpoint inhibitors can add to the arsenal of strategies in the fight against cancer.Although RANK and RANKL were first identified as critical players in bone remodeling, crucial roles of this pathway in controlling key aspects of the immune system and cancer as well as integrating sex hormone signaling to physiological adaptations have been uncovered and dissected in recent years. In the mammary gland, RANKL, induced by progesterone, signals to hormone receptor-negative RANK expressing epithelial cells to stimulate mammary progenitor cell expansion. Recently we and other groups have provided direct genetic and pharmacological proof that the RANKL/RANK pathway plays an essential role in the progression of familial JM, SC, and JP contributed to writing this review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Facial diplegia, a rare variant of Guillain-Barré syndrome (GBS), is a challenging diagnosis to make in the emergency department due to its resemblance to neurologic Lyme disease.We present a case of a 27-year-old previously healthy man who presented with bilateral facial paralysis.Despite the variance in presentation, the recommended standard of practice for diagnostics  and disposition  of facial diplegia are the same as for other presentations of GBS.When presented with bilateral facial palsy emergency providers should consider autoimmune, infectious, idiopathic, metabolic, neoplastic, neurologic, and traumatic etiologies in addition to the much more common neurologic Lyme disease. Facial diplegia is a rare variant of Guillain-Barré syndrome (GBS) where patients present with bilateral facial paralysis and paresthesia. Patients usually present 1–28 days after onset of symptoms, occasionally with extremity paresthesia but often in isolation.A 27-year-old male military recruit without significant previous medical history was transported to the emergency department (ED) by ambulance with a chief complaint of bilateral facial paralysis. A resident of Puerto Rico, the patient had recently traveled to San Antonio, Texas, for military exercises and received multiple vaccines five days prior to onset of symptoms. He began experiencing mild paresthesia in his hands and feet four days prior to ED presentation. He reported progressive neurologic signs and symptoms including being unable to close his eyes or mouth, which made sleeping and eating difficult, as well as right-sided facial numbness. He went to the military medical clinic two days prior to ED presentation where he was diagnosed with a complex migraine and discharged to the barracks on quarters (strict bed rest) with ibuprofen and ondansetron and instructions to return for follow-up the next day to evaluate for resolution of symptoms.During the subsequent ED visit his vital signs included a heart rate of 113 beats per minute, blood pressure 117/76 millimeters of mercury, respiration 16 breaths per minute, and temperature 98.5 degrees Fahrenheit. His blood glucose was 116 milligrams per deciliter (mg/dL) (90–120 mg/dL). Physical exam revealed symmetric bilateral facial paralysis, causing difficulty in closing his eyelids and mouth, which resulted in injected conjunctiva with moderate tearing, and cheilitis . He endoComputed tomography of his head showed no masses or evidence of bleeding. Neurology was consulted and per their recommendation we obtained magnetic resonance imaging of his brain, which demonstrated no lesions, masses, or evidence of edema. A lumbar puncture was performed; it revealed albumin-cytological dissociation of cerebrospinal fluid (CSF), which is an increased protein with normal cell counts. Polymerase chain reaction did not detect any common bacterial or viral meningitis organisms. His complete blood count, comprehensive metabolic panel, and erythrocyte sedimentation rate were all within normal limits. The patient was given 10 milligrams dexamethasone intravenously in the ED, and blood cultures were obtained prior to admission.What do we already know about this clinical entity?The treatment and disposition for all variants of Guillain-Barré syndrome (GBS) is the same.What makes this presentation of disease reportable?Facial diplegia is a rare variant of GBS that mimics a fairly common disease process in Lyme diseaseWhat is the major learning point?This case demonstrates the differential diagnosis and diagnostic pitfalls associated with facial diplegia.How might this improve emergency medicine practice?Clinicians should consider a thorough differential diagnosis for facial diplegia.Borrelia burgdorferi, Zika virus, West Nile virus, varicella-zoster virus, Treponema pallidum, human immunodeficiency virus, or Mycobacterium tuberculosis. Based upon the exam findings and diagnostic testing, the diagnosis of facial diplegia (GBS variant) was confirmed with the etiology most likely post-vaccinal. The patient received four days of IVIG and was discharged from the hospital. He returned to training with 4/5 strength and decreased light touch in his bilateral face and complete resolution of the paresthesia in his extremities. He was started on long-term daily corticosteroids by the neurologist with continued 4/5 strength and light touch in his bilateral face noted on his one month follow-up.With a presumptive diagnosis of GBS the patient was admitted to the hospital for serial negative inspiratory flow measurements and intravenous immune globulin (IVIG), while awaiting further diagnostic testing by neurology. Serum and CSF studies detected none of the following organisms: Facial diplegia is a rare disorder, accounting for less than 2% of all patients with facial paralysis.4Common features of GBS include the following: subjective peripheral paresthesia (88%); a preceding illness one to two weeks prior to symptoms 75%); and CSF albumin-cytological dissociation (88%).%; and CSThe prognosis for the facial diplegia subtype of GBS is generally favorable, as diaphragmatic paralysis has never been documented.2Facial diplegia is a rare variant of GBS that presents a very challenging diagnosis to make in the ED due to its wide differential diagnosis. When presented with bilateral facial palsy emergency providers should consider autoimmune, infectious, idiopathic, metabolic, neoplastic, neurologic, and traumatic etiologies in addition to the much more common neurologic Lyme disease. Facial diplegia is a clinical diagnosis that is bolstered by the presence of albumin-cytological dissociation in the cerebral spinal fluid.