The Cytoskeleton as Regulator of Cell Signaling Pathways.

During interphase, filamentous actin, microtubules, and intermediate filaments regulate cell shape, motility, transport, and interactions with the environment. These activities rely on signaling events that control cytoskeleton properties. Recent studies uncovered mechanisms that go far beyond this one-directional flow of information. Thus, the three branches of the cytoskeleton impinge on signaling pathways to determine their activities. We propose that this regulatory role of the cytoskeleton provides sophisticated mechanisms to control the spatiotemporal output and the intensity of signaling events. Specific examples emphasize these emerging contributions of the cytoskeleton to cell physiology. In our opinion, further exploration of these pathways will uncover new concepts of cellular communication that originate from the cytoskeleton.

Yeast profilin mutants inhibit classical nuclear import and alter the balance between actin and tubulin levels.

Profilin is a small protein that controls actin polymerization in yeast and higher eukaryotes. In addition, profilin has emerged as a multifunctional protein that contributes to other processes in multicellular organisms. This study focuses on profilin (Pfy1) in the budding yeast Saccharomyces cerevisiae. The primary sequences of yeast Pfy1 and its metazoan orthologs diverge vastly. However, structural elements of profilin are conserved among different species. To date, the full spectrum of Pfy1 functions has yet to be defined. The current work explores the possible involvement of yeast profilin in nuclear protein import. To this end, a panel of well-characterized yeast profilin mutants was evaluated. The experiments demonstrate that yeast profilin (i) regulates nuclear protein import, (ii) determines the subcellular localization of essential nuclear transport factors, and (iii) controls the relative abundance of actin and tubulin. Together, these results define yeast profilin as a moonlighting protein that engages in multiple essential cellular activities.

Mitochondrial Oxidative Stress Reduces the Immunopotency of Mesenchymal Stromal Cells in Adults With Coronary Artery Disease.

RATIONALE: Mesenchymal stromal cells (MSCs) are promising therapeutic strategies for coronary artery disease; however, donor-related variability in cell quality is a main cause of discrepancies in preclinical studies. In vitro, MSCs from individuals with coronary artery disease have reduced ability to suppress activated T-cells. The mechanisms underlying the altered immunomodulatory capacity of MSCs in the context of atherosclerosis remain elusive. OBJECTIVE: The aim of this study was to assess the role of mitochondrial dysfunction in the impaired immunomodulatory properties of MSCs from patients with atherosclerosis. METHODS AND RESULTS: Adipose tissue-derived MSCs were isolated from atherosclerotic (n=38) and nonatherosclerotic (n=42) donors. MSCs:CD4+T-cell suppression was assessed in allogeneic coculture systems. Compared with nonatherosclerotic-MSCs, atherosclerotic-MSCs displayed higher levels of both intracellular (P=0.006) and mitochondrial (P=0.03) reactive oxygen species reflecting altered mitochondrial function. The increased mitochondrial reactive oxygen species levels of atherosclerotic-MSCs promoted a phenotypic switch characterized by enhanced glycolysis and an altered cytokine secretion (interleukin-6 P<0.0001, interleukin-8/C-X-C motif chemokine ligand 8 P=0.04, and monocyte chemoattractant protein-1/chemokine ligand 2 P=0.01). Furthermore, treatment of atherosclerotic-MSCs with the reactive oxygen species scavenger N-acetyl-l-cysteine reduced the levels of interleukin-6, interleukin-8/C-X-C motif chemokine ligand 8, and monocyte chemoattractant protein-1/chemokine ligand 2 in the MSC secretome and improved MSCs immunosuppressive capacity (P=0.03). CONCLUSIONS: An impaired mitochondrial function of atherosclerotic-MSCs underlies their altered secretome and reduced immunopotency. Interventions aimed at restoring the mitochondrial function of atherosclerotic-MSCs improve their in vitro immunosuppressive ability and may translate into enhanced therapeutic efficiency.

Cellular senescence is associated with reorganization of the microtubule cytoskeleton.

Senescent cells undergo structural and functional changes that affect essentially every aspect of cell physiology. To date, the impact of senescence on the cytoskeleton is poorly understood. This study evaluated the cytoskeleton in two independent cellular models of kidney epithelium senescence. Our work identified multiple senescence-related alterations that impact microtubules and filamentous actin during interphase. Both filamentous systems reorganized profoundly when cells became senescent. As such, microtubule stability increased during senescence, making these filaments more resistant to disassembly in the cold or by nocodazole. Microtubule stabilization was accompanied by enhanced α-tubulin acetylation on lysine 40 and the depletion of HDAC6, the major deacetylase for α-tubulin lysine 40. Rho-associated kinase Rock1 is an upstream regulator that modulates key properties of the cytoplasmic cytoskeleton. Our research shows that Rock1 concentrations were reduced significantly in senescent cells, and we revealed a mechanistic link between microtubule stabilization and Rock1 depletion. Thus, Rock1 overexpression partially restored the cold sensitivity of microtubules in cells undergoing senescence. Additional components relevant to microtubules were affected by senescence. Specifically, we uncovered the senescence-related loss of the microtubule nucleating protein γ-tubulin and aberrant formation of γ-tubulin foci. Concomitant with the alterations of microtubule and actin filaments, senescent cells displayed functional changes. In particular, cell migration was impaired significantly in senescent cells. Taken together, our study identified new senescence-associated deficiencies of the microtubule and actin cytoskeleton, provided insights into the underlying molecular mechanisms and demonstrated functional consequences that are important to the physiology and function of renal epithelial cells.

Gold nanourchins induce cellular stress, impair proteostasis and damage RNA.

Gold nanoparticles have excellent potential for theranostic applications, but their impact on living cells is only partially understood. Many gold nanoparticles enter cells through endosomes/lysosomes which are linked to different cell organelles and compartments. Our study focuses on the unfolded protein response (UPR) in the endoplasmic reticulum (ER), cytoplasmic RNA-granules and proteostasis, because they are established indicators of cell stress and key regulators of cellular homeostasis. Using HeLa and renal proximal tubule cells as model systems, we show that gold nanourchins reduce cell proliferation, cause ER stress and impair proteostasis. Specifically, gold nanourchins activate the PERK-branch of the UPR, promote RNA oxidation, enhance P-body formation, and accumulate the oxidative stress marker Nrf2 and NFκB in nuclei. Taken together, our study demonstrates that gold nanourchins compromise ER, redox, protein, and RNA homeostasis. These insights provide new information on the cellular responses and molecular changes that gold nanourchins elicit in mammalian cells.

Dissecting the molecular mechanisms that impair stress granule formation in aging cells.

Aging affects numerous aspects of cell biology, but the senescence-associated changes in the stress response are only beginning to emerge. To obtain mechanistic insights into these events, we examined the formation of canonical and non-canonical stress granules (SGs) in the cytoplasm. SG generation is a key event after exposure to physiological or environmental stressors. It requires the SG-nucleating proteins G3BP1 and TIA-1/TIAR and stress-related signaling events. To analyze SG formation, we used two independent models of somatic cell aging. In both model systems, cellular senescence impaired the assembly of two SG classes: (i) it compromised the formation of canonical SGs, and (ii) skewed the production of non-canonical SGs. We dissected the mechanisms underlying these senescence-dependent changes in granule biogenesis and identified several specific targets that were modulated by aging. Thus, we demonstrate a depletion of G3BP1 and TIA-1/TIAR in senescent cells and show that the loss of G3BP1 contributed to impaired SG formation. We further reveal that aging reduced Sp1 levels; this transcription factor regulated G3BP1 and TIA-1/TIAR abundance. The assembly of canonical SGs relies on the phosphorylation of translation initiation factor eIF2α. We show that senescence can cause eIF2α hyperphosphorylation. CReP is a subunit of protein phosphatase 1 and critical to reverse the stress-dependent phosphorylation of eIF2α. We demonstrate that the loss of CReP correlated with the aging-related hyperphosphorylation of eIF2α. Together, we have identified significant changes in the stress response of aging cells and provide mechanistic insights. Based on our work, we propose that the decline in SG formation can provide a new biomarker to evaluate cellular aging.

Cytoplasmic RNA Granules in Somatic Maintenance.

Cytoplasmic RNA granules represent subcellular compartments that are enriched in protein-bound RNA species. RNA granules are produced by evolutionary divergent eukaryotes, including yeast, mammals, and plants. The functions of cytoplasmic RNA granules differ widely. They are dictated by the cell type and physiological state, which in turn is determined by intrinsic cell properties and environmental factors. RNA granules provide diverse cellular functions. However, all of the granules contribute to aspects of RNA metabolism. This is exemplified by transcription, RNA storage, silencing, and degradation, as well as mRNP remodeling and regulated translation. Several forms of cytoplasmic mRNA granules are linked to normal physiological processes. For instance, they may coordinate protein synthesis and thereby serve as posttranscriptional "operons". RNA granules also participate in cytoplasmic mRNA trafficking, a process particularly well understood for neurons. Many forms of RNA granules support the preservation of somatic cell performance under normal and stress conditions. On the other hand, severe insults or disease can cause the formation and persistence of RNA granules that contribute to cellular dysfunction, especially in the nervous system. Neurodegeneration and many other diseases linked to RNA granules are associated with aging. Nevertheless, information related to the impact of aging on the various types of RNA granules is presently very limited. This review concentrates on cytoplasmic RNA granules and their role in somatic cell maintenance. We summarize the current knowledge on different types of RNA granules in the cytoplasm, their assembly and function under normal, stress, or disease conditions. Specifically, we discuss processing bodies, neuronal granules, stress granules, and other less characterized cytoplasmic RNA granules. Our focus is primarily on mammalian and yeast models, because they have been critical to unravel the physiological role of various RNA granules. RNA granules in plants and pathogens are briefly described. We conclude our viewpoint by summarizing the emerging concepts for RNA granule biology and the open questions that need to be addressed in future studies.

Cytoplasmic stress granules: Dynamic modulators of cell signaling and disease.

Stress granule (SG) assembly is a conserved cellular strategy to minimize stress-related damage and promote cell survival. Beyond their fundamental role in the stress response, SGs have emerged as key players for human health. As such, SG assembly is associated with cancer, neurodegenerative disorders, ischemia, and virus infections. SGs and granule-related signaling circuits are therefore promising targets to improve therapeutic intervention for several diseases. This is clinically relevant, because pharmacological drugs can affect treatment outcome by modulating SG formation. As membraneless and highly dynamic compartments, SGs regulate translation, ribostasis and proteostasis. Moreover, they serve as signaling hubs that determine cell viability and stress recovery. Various compounds can modulate SG formation and dynamics. Rewiring cell signaling through SG manipulation thus represents a new strategy to control cell fate under various physiological and pathological conditions.

Impact of Leishmania infection on host macrophage nuclear physiology and nucleopore complex integrity.

The protease GP63 is an important virulence factor of Leishmania parasites. We previously showed that GP63 reaches the perinuclear area of host macrophages and that it directly modifies nuclear translocation of the transcription factors NF-κB and AP-1. Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes. Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations. In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation. Collectively, our data indicates that Leishmania infection strongly affects nuclear physiology, suggesting that targeting of nuclear physiology may be a strategy beneficial for virulent Leishmania parasites.

Nucleoli and stress granules: connecting distant relatives.

Nucleoli and cytoplasmic stress granules (SGs) are subcellular compartments that modulate the response to endogenous and environmental signals to control cell survival. In our opinion, nucleoli and SGs are functionally linked; they are distant relatives that combine forces when cellular homeostasis is threatened. Several lines of evidence support this idea; nucleoli and SGs share molecular building blocks, are regulated by common signaling pathways and communicate when vital cellular functions become compromised. Together, nucleoli and SGs orchestrate physiological responses that are directly relevant to stress and human health. As both compartments have established roles in neurodegenerative diseases, cancer and virus infections, we propose that these conditions will benefit from therapeutic interventions that target simultaneously nucleoli and SGs.

AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules.

5'-AMP-activated protein kinase alpha regulates stress granule biogenesis.

Stress granule (SG) assembly represents a conserved eukaryotic defense strategy against various insults. Although essential for the ability to cope with deleterious conditions, the signaling pathways controlling SG formation are not fully understood. The energy sensor AMP-activated protein kinase (AMPK) is critical for the cellular stress response. Human cells produce two AMPK catalytic α-subunits with not only partially overlapping, but also unique functions. Here, we provide direct support for structural and functional links between AMPK-α isoforms and SGs. As such, several stressors promote SG association of AMPK-α2, but not AMPK-α1. Multiple lines of evidence link AMPK activity to SG biogenesis. First, pharmacological kinase inhibition interfered with SG formation. Second, AMPK-α knockdown combined with in-depth quantitative SG analysis revealed isoform-specific changes of SG characteristics. Third, overexpression of mutant α-subunits further substantiated that AMPK regulates SG parameters. Finally, we identified the SG-nucleating protein G3BP1 as an AMPK-α2 binding partner. This interaction is stimulated by stress and notably occurs in SGs. Collectively, our data define the master metabolic regulator AMPK as a novel SG constituent that also controls their biogenesis.

Consistent sex-dependent effects of PKMζ gene ablation and pharmacological inhibition on the maintenance of referred pain.

BACKGROUND: Persistently active PKMζ has been implicated in maintaining spinal nociceptive sensitization that underlies pain hypersensitivity. However, evidence for PKMζ in the maintenance of pain hypersensitivity comes exclusively from short-term studies in males using pharmacological agents of questionable selectivity. The present study examines the contribution of PKMζ to long-lasting allodynia associated with neuropathic, inflammatory, or referred visceral and muscle pain in males and females using pharmacological inhibition or genetic ablation. RESULTS: Pharmacological inhibition or genetic ablation of PKMζ reduced mild formalin pain and slowly developing contralateral allodynia in nerve-injured rats, but not moderate formalin pain or ipsilateral allodynia in models of neuropathic and inflammatory pain. Pharmacological inhibition or genetic ablation of PKMζ also effectively reduced referred visceral and muscle pain in male, but not in female mice and rats. CONCLUSION: We show pharmacological inhibition and genetic ablation of PKMζ consistently attenuate long-lasting pain hypersensitivity. However, differential effects in models of referred versus inflammatory and neuropathic pain, and in males versus females, highlight the roles of afferent input-dependent masking and sex differences in the maintenance of pain hypersensitivity.

Gold nanoparticles induce nuclear damage in breast cancer cells, which is further amplified by hyperthermia.

Gold nanoparticles have emerged as promising tools for cancer research and therapy, where they can promote thermal killing. The molecular mechanisms underlying these events are not fully understood. The geometry and size of gold nanoparticles can determine the severity of cellular damage. Therefore, small and big gold nanospheres as well as gold nanoflowers were evaluated side-by-side. To obtain quantitative data at the subcellular and molecular level, we assessed how gold nanoparticles, either alone or in combination with mild hyperthermia, altered the physiology of cultured human breast cancer cells. Our analyses focused on the nucleus, because this organelle is essential for cell survival. We showed that all the examined gold nanoparticles associated with nuclei. However, their biological effects were quantitatively different. Thus, depending on the shape and size, gold nanoparticles changed multiple nuclear parameters. They redistributed stress-sensitive regulators of nuclear biology, altered the nuclear morphology, reorganized nuclear laminae and envelopes, and inhibited nucleolar functions. In particular, gold nanoparticles reduced the de novo biosynthesis of RNA in nucleoli, the subnuclear compartments that produce ribosomes. While small gold nanospheres and nanoflowers, but not big gold nanospheres, damaged the nucleus at normal growth temperature, several of these defects were further exacerbated by mild hyperthermia. Taken together, the toxicity of gold nanoparticles correlated with changes in nuclear organization and function. These results emphasize that the cell nucleus is a prominent target for gold nanoparticles of different morphologies. Moreover, we demonstrated that RNA synthesis in nucleoli provides quantitative information on nuclear damage and cancer cell survival.

Chaperones and multitasking proteins in the nucleolus: networking together for survival?

The nucleolus has emerged as a key player that regulates cell growth, survival and the recovery from stress. Progress in proteomics made it possible to sequence the nucleolar proteome under different physiological conditions. Together with other research, this work revealed the presence of multiple chaperones and co-chaperones in the nucleolus. Molecular chaperones are components of a larger network that promotes protein homeostasis, thereby providing continuous adaptation to a changing environment. Recent studies suggest that the cellular chaperone network is divided into individual branches which orchestrate specific functions. Input from separate branches is then combined to 'fine-tune' the cellular proteostasis network. Based on the latest developments in nucleolar and chaperone biology, we speculate that a unique network comprising chaperones, co-chaperones and multitasking proteins is located in nucleoli. This network supports and regulates fundamental biological processes, including ribosome biogenesis, cell signaling, and the stress response.

Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization.

The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.

Oxidative stress and signaling through EGFR and PKA pathways converge on the nuclear transport factor RanBP1.

Nuclear protein trafficking requires the soluble transport factor RanBP1. The subcellular distribution of RanBP1 is dynamic, as the protein shuttles between the nucleus and cytoplasm. To date, the signaling pathways regulating RanBP1 subcellular localization are poorly understood. During interphase, RanBP1 resides mostly in the cytoplasm. We show here that oxidative stress concentrates RanBP1 in the nucleus, and our study defines the underlying mechanisms. Specifically, RanBP1's cysteine residues are not essential for its oxidant-induced relocation. Furthermore, our pharmacological approaches uncover that signaling mediated by epidermal growth factor receptor (EGFR) and protein kinase A (PKA) control RanBP1 localization during stress. In particular, pharmacological inhibitors of EGFR or PKA diminish the oxidant-dependent relocation of RanBP1. Mutant analysis identified serine 60 and tyrosine 103 as regulators of RanBP1 nuclear accumulation during oxidant exposure. Taken together, our results define RanBP1 as a target of oxidative stress and a downstream effector of EGFR and PKA signaling routes. This positions RanBP1 at the intersection of important cellular signaling circuits.

Pifithrin-µ Induces Stress Granule Formation, Regulates Cell Survival, and Rewires Cellular Signaling.

(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5'-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds.

Silica-coated LiYF4:Yb3+, Tm3+ upconverting nanoparticles are non-toxic and activate minor stress responses in mammalian cells.

Lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates for use in biomedicine. The interaction of nanomaterials with biological systems determines whether they are suitable for use in living cells. In-depth knowledge of the nano-bio interactions is therefore a pre-requisite for the development of biomedical applications. The current study evaluates fundamental aspects of the NP-cell interface for square bipyramidal UCNPs containing a LiYF4:Yb3+, Tm3+ core and two different silica surface coatings. Given their importance for mammalian physiology, fibroblast and renal proximal tubule epithelial cells were selected as cellular model systems. We have assessed the toxicity of the UCNPs and measured their impact on the homeostasis of living non-malignant cells. Rigorous analyses were conducted to identify possible toxic and sub-lethal effects of the UCNPs. To this end, we examined biomarkers that reveal if UCNPs induce cell killing or stress. Quantitative measurements demonstrate that short-term exposure to the UCNPs had no profound effects on cell viability, cell size or morphology. Indicators of oxidative, endoplasmic reticulum, or nucleolar stress, and the production of molecular chaperones varied with the surface modification of the UCNPs and the cell type analyzed. These differences emphasize the importance of evaluating cells of diverse origin that are relevant to the intended use of the nanomaterials. Taken together, we established that short-term, our square bipyramidal UCNPs are not toxic to non-malignant fibroblast and proximal renal epithelial cells. Compared with established inducers of cellular stress, these UCNPs have minor effects on cellular homeostasis. Our results build the foundation to explore square bipyramidal UCNPs for future in vivo applications.

Automated detection and quantification of granular cell compartments.

Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules (SGs). To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming, while preventing the efficient use of high-throughput technology. We developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Our methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of our protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, we show that the type of stress determines granule size and composition, as illustrated by the concentration of poly(A)-RNA and a specific SG marker protein. Furthermore, we took advantage of the computer-based and automated methods to design assays suitable for high-throughput screening.