A cell-based assay for rapid assessment of ACE2 catalytic function.

Angiotensin-converting enzyme II (ACE2) is a monocarboxypeptidase expressed throughout multiple tissues and its catalysis of bioactive peptides regulates the renin-angiotensin system mediating blood pressure homeostasis. ACE2 is implicated in a variety of diseases, including obesity, diabetes, and cardiovascular diseases, and is the obligate entry receptor for SARS-CoV-2 infection. Disease-associated genetic variants of ACE2 are increasingly being identified but are poorly characterized. To aid this problem, we introduce a fluorometric cell-based assay for evaluating surface-expressed ACE2 catalytic activity that preserves the native glycosylation of the host environment and is amenable to high-throughput analysis of ACE2 variants in multi-well plates. We demonstrate sensitivity to detecting catalysis of the key ACE2 substrates, Angiotensin II, Apelin-13, and des-Arg9-bradykinin, and impact of a catalytically-deficient ACE2 variant. Normalizing catalytic measures to surface ACE2 expression accounts for variability in ACE2 variant transfection, surface delivery or stability. This assay provides a convenient and powerful approach for investigating the catalytic characteristics of ACE2 variants involved in cardiovascular peptide cascades and homeostasis of multiple organs.

Multi-parametric analysis of 57 SYNGAP1 variants reveal impacts on GTPase signaling, localization, and protein stability.

SYNGAP1 is a neuronal Ras and Rap GTPase-activating protein with important roles in regulating excitatory synaptic plasticity. While many SYNGAP1 missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia, and autism spectrum disorder (ASD), whether and how they contribute to individual disease phenotypes is often unknown. Here, we characterize 57 variants in seven assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure variant impact on protein stability, pERK, pGSK3ß, pp38, pCREB, and high-content imaging to examine subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wild-type (WT)-like in their regulation of pERK and pGSK3ß, while all variants retain at least partial ability to dephosphorylate pCREB. Interestingly, our assays reveal that a larger proportion of variants located within the disordered domain of unknown function (DUF) comprising the C-terminal half of SYNGAP1 exhibited higher LoF, compared to variants within the better studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction for only two missense variants, both located within the catalytic domain. Using high-content imaging, we find variants located within the C2 domain known to mediate membrane lipid interactions exhibit significantly larger cytoplasmic speckles than WT SYNGAP1. Moreover, this subcellular phenotype shows both correlation with altered catalytic activity and unique deviation from signaling assay results, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence, multi-functional measures for making pathogenicity predictions.

Cx43 in Neural Progenitors Promotes Glioma Invasion in a 3D Culture System.

The environment that envelops the cancer cells intimately affects the malignancy of human cancers. In the case of glioma, an aggressive adult brain cancer, its high rate of recurrence after total resection is responsible for a poor prognosis. Connexin43 (Cx43) is a gap junction protein with a prominent presence in glioma-associated normal brain cells, specifically in the reactive astrocytes. We previously demonstrated that elimination of Cx43 in these astrocytes reduces glioma invasion in a syngeneic mouse model. To further our investigation in human glioma cells, we developed a scaffold-free 3D platform that takes into account both the tumor and its interaction with the surrounding tissue. Using cell-tracking dyes and 3D laser scanning confocal microscopy, we now report that the elimination of Cx43 protein in neural progenitor spheroids reduced the invasiveness of human brain tumor-initiating cells, confirming our earlier observation in an intact mouse brain. By investigating the glioma invasion in a defined multicellular system with a tumor boundary that mimics the intact brain environment, our findings strengthen Cx43 as a candidate target for glioma control.

Na/H exchanger NHE1 acts upstream of rho GTPases to promote neurite outgrowth.

Na+/H+ exchanger NHE1, a major determinant of intracellular pH (pHi) in mammalian central neurons, promotes neurite outgrowth under both basal and netrin-1-stimulated conditions. The small GTP binding proteins and their effectors have a dominant role in netrin-1-stimulated neurite outgrowth. Since NHE1 has been shown previously to work downstream of the Rho GTPases-mediated polarized membrane protrusion in non-neuronal cells, we examined whether NHE1 has a similar relationship with Cdc42, Rac1 and RhoA in neuronal morphogenesis. Interestingly, our results suggest the possibility that NHE1 acting upstream of Rho GTPases to promote neurite outgrowth induced by netrin-1. First, we found that netrin-1-induced increases in the activities of Rho GTPases using FRET (Forster Resonance Energy Transfer) analyses in individual growth cones; furthermore, their increased activities were abolished by cariporide, a specific NHE1 inhibitor. Second, NHE1 inhibition had no effect on neurite retraction induced by L-α-Lysophosphatidic acid (LPA), a potent RhoA activator. The regulation of Rho GTPases by NHE1 was further evidenced by reduced Rac1, Cdc42 and RhoA activities in NHE1-null neurons. Taken together, our findings suggest that NHE1-dependent neuronal morphogenesis involves the activation of Rho-family of small GTPases.

Connexin43 peptide, TAT-Cx43266-283, selectively targets glioma cells, impairs malignant growth, and enhances survival in mouse models in vivo.

BACKGROUND: Malignant gliomas are the most frequent primary brain tumors and remain among the most incurable cancers. Although the role of the gap junction protein, connexin43 (Cx43), has been deeply investigated in malignant gliomas, no compounds have been reported with the ability to recapitulate the tumor suppressor properties of this protein in in vivo glioma models. METHODS: TAT-Cx43266-283 a cell-penetrating peptide which mimics the effect of Cx43 on c-Src inhibition, was studied in orthotopic immunocompetent and immunosuppressed models of glioma. The effects of this peptide in brain cells were also analyzed. RESULTS: While glioma stem cell malignant features were strongly affected by TAT-Cx43266-283, these properties were not significantly modified in neurons and astrocytes. Intraperitoneally administered TAT-Cx43266-283 decreased the invasion of intracranial tumors generated by GL261 mouse glioma cells in immunocompetent mice. When human glioma stem cells were intracranially injected with TAT-Cx43266-283 into immunodeficient mice, there was reduced expression of the stemness markers nestin and Sox2 in human glioma cells at 7 days post-implantation. Consistent with the role of Sox2 as a transcription factor required for tumorigenicity, TAT-Cx43266-283 reduced the number and stemness of human glioma cells at 30 days post-implantation. Furthermore, TAT-Cx43266-283 enhanced the survival of immunocompetent mice bearing gliomas derived from murine glioma stem cells. CONCLUSION: TAT-Cx43266-283 reduces the growth, invasion, and progression of malignant gliomas and enhances the survival of glioma-bearing mice without exerting toxicity in endogenous brain cells, which suggests that this peptide could be considered as a new clinical therapy for high-grade gliomas.

Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation.

Extracellular matrix (ECM) remodeling, degradation and glioma cell motility are critical aspects of glioblastoma multiforme (GBM). Despite being a rich source of potential biomarkers and targets for therapeutic advance, the dynamic changes occurring within the extracellular environment that are specific to GBM motility have yet to be fully resolved. The gap junction protein connexin43 (Cx43) increases glioma migration and invasion in a variety of in vitro and in vivo models. In this study, the upregulation of Cx43 in C6 glioma cells induced morphological changes and the secretion of proteins associated with cell motility. Demonstrating the selective engagement of ECM remodeling networks, secretome analysis revealed the near-binary increase of osteopontin and matrix metalloproteinase-3 (MMP3), with gelatinase and NFF-3 assays confirming the proteolytic activities. Informatic analysis of interactome and secretome downstream of Cx43 identifies networks of glioma motility that appear to be synergistically engaged. The data presented here implicate ECM remodeling and matrikine signals downstream of Cx43/MMP3/osteopontin and ARK1B10 inhibition as possible avenues to inhibit GBM.

GJA1 (connexin43) is a key regulator of Alzheimer's disease pathogenesis.

GJA1 (connexin43) has been predicted as the top key driver of an astrocyte enriched subnetwork associated with Alzheimer's disease (AD). In this study, we comprehensively examined GJA1 expression across 29 transcriptomic and proteomic datasets from post-mortem AD and normal control brains. We demonstrated that GJA1 was strongly associated with AD amyloid and tau pathologies and cognitive functions. RNA sequencing analysis of Gja1-/- astrocytes validated that Gja1 regulated the subnetwork identified in AD, and many genes involved in Aß metabolism. Astrocytes lacking Gja1 showed reduced Apoe protein levels as well as impaired Aß phagocytosis. Consistent with this, wildtype neurons co-cultured with Gja1-/- astrocytes contained higher levels of Aß species than those with wildtype astrocytes. Moreover, Gja1-/- astrocytes was more neuroprotective under Aß stress. Our results underscore the importance of GJA1 in AD pathogenesis and its potential for further investigation as a promising pharmacological target in AD.

Modelling glioma invasion using 3D bioprinting and scaffold-free 3D culture.

Glioma is a highly aggressive form of brain cancer, with some subtypes having 5-year survival rates of less than 5%. Tumour cell invasion into the surrounding parenchyma seems to be the primary driver of these poor outcomes, as most gliomas recur within 2 cm of the original surgically-resected tumour. Many current approaches to the development of anticancer therapy attempt to target genetic weaknesses in a particular cancer, but may not take into account the microenvironment experienced by a tumour and the patient-specific genetic differences in susceptibility to treatment. Here we demonstrate the use of complementary approaches, 3D bioprinting and scaffold-free 3D tissue culture, to examine the invasion of glioma cells into neural-like tissue with 3D confocal microscopy. We found that, while both approaches were successful, the use of 3D tissue culture for organoid development offers the advantage of broad accessibility. As a proof-of-concept of our approach, we developed a system in which we could model the invasion of human glioma cells into mouse neural progenitor cell-derived spheroids. We show that we can follow invasion of human tumour cells using cell-tracking dyes and 3D laser scanning confocal microscopy, both in real time and in fixed samples. We validated these results using conventional cryosectioning. Our scaffold-free 3D approach has broad applicability, as we were easily able to examine invasion using different neural progenitor cell lines, thus mimicking differences that might be observed in patient brain tissue. These results, once applied to iPSC-derived cerebral organoids that incorporate the somatic genetic variability of patients, offer the promise of truly personalized treatments for brain cancer.

Novel approach to temozolomide resistance in malignant glioma: connexin43-directed therapeutics.

Resistance of malignant glioma, including glioblastoma (GBM), to the chemotherapeutic temozolomide (TMZ) remains a key obstacle in treatment strategies. The gap junction protein connexin43 (Cx43) has complex roles in the establishment, progression, and persistence of malignant glioma. Recent findings demonstrate that connexins play an important role in the microenvironment of malignant glioma and that Cx43 is capable of conferring chemotherapeutic resistance to GBM cells. Carboxyl-terminal Cx43 peptidomimetics show therapeutic promise in overcoming TMZ resistance via mechanisms that may include modulating junctional activity between tumor cells and peritumoral cells and/or downstream molecular signaling events mediated by Cx43 protein binding. High levels of intra-tumor and inter-tumor heterogeneity make it difficult to clearly define specific populations for Cx43-targeted therapy; hence, development of in vitro models that better mimic the microenvironment of malignant glioma, and the incorporation of patient-derived stem cells, could provide opportunities for patient-specific drug screening. This review summarizes recent advances in understanding the roles of Cx43 in malignant glioma, with a special focus on tumor microenvironment, TMZ resistance, and therapeutic opportunity offered by Cx43 peptidomimetics.

Conflicting Roles of Connexin43 in Tumor Invasion and Growth in the Central Nervous System.

The tumor microenvironment is known to have increased levels of cytokines and metabolites, such as glutamate, due to their release from the surrounding cells. A normal cell around the tumor that responds to the inflammatory environment is likely to be subsequently altered. We discuss how these abnormalities will support tumor survival via the actions of gap junctions (GJs) and hemichannels (HCs) which are composed of hexamer of connexin43 (Cx43) protein. In particular, we discuss how GJ intercellular communication (GJIC) in glioma cells, the primary brain tumor, is a regulatory factor and its attenuation leads to tumor invasion. In contrast, the astrocytes, which are normal cells around the glioma, are "hijacked" by tumor cells, either by receiving the transmission of malignant substances from the cancer cells via GJIC, or perhaps via astrocytic HC activity through the paracrine signaling which enable the delivery of these substances to the distal astrocytes. This astrocytic signaling would promote tumor expansion in the brain. In addition, brain metastasis from peripheral tissues has also been known to be facilitated by GJs formed between cerebral vascular endothelial cells and cancer cells. Astrocytes and microglia are generally thought to eliminate cancer cells at the blood-brain barrier. In contrast, some reports suggest they facilitate tumor progression as tumor cells take advantage of the normal functions of astrocytes that support the survival of the neurons by exchanging nutrients and metabolites. In summary, GJIC is essential for the normal physiological function of growth and allowing the diffusion of physiological substances. Therefore, whether GJIC is cancer promoting or suppressing may be dependent on what permeates through GJs, when it is active, and to which cells. The nature of GJs, which has been ambiguous in brain tumor progression, needs to be revisited and understood together with new findings on Cx proteins and HC activities.

An update on minding the gap in cancer.

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below.

Acute connexin43 temporal and spatial expression in response to ischemic stroke.

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.

Common mechanisms linking connexin43 to neural progenitor cell migration and glioma invasion.

Cell migration is critical for cell differentiation, tissue formation and organ development. Several mechanisms come to play in the process of cell migration, orchestrating changes in cell polarity, adhesion, process extension and motility. Recent findings have shown that gap junctions, and specifically connexin43 (Cx43), can play a significant role in these processes, impacting adhesion and cytoskeletal rearrangements. Thus Cx43 within a cell regulates its motility and migration via intracellular signaling. Furthermore, Cx43 in the host cells can impact the degree of cellular migration through that tissue. Similarities in these connexin-based processes account for both neural progenitor migration in the developing brain, and for glioma cell invasion in the mature brain. In both cases, Cx43 in the tissue ("soil") in which cells ("seeds") exist facilitates their migration and, for glioma cells, tissue invasion. Cx43 mediates these effects through channel- and non-channel-dependent mechanisms which have similarities in both paradigms of cell migration. This provides insight into developmental processes and pathological situations, as well as possible therapeutic approaches regarding specific functional domains of gap junction proteins.

Gap junctions modulate glioma invasion by direct transfer of microRNA.

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.

Reduction in gap junction intercellular communication promotes glioma migration.

Glioblastoma Multiforme (GBM), an aggressive form of adult brain tumor, is difficult to treat due to its invasive nature. One of the molecular changes observed in GBM is a decrease in the expression of the gap junction protein Connexin43 (Cx43); however, how a reduction in Cx43 expression contributes to glioma malignancy is still unclear. In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity. We used a 3D spheroid migration model that mimics the in vivo architecture of tumor cells to quantify migration changes. We found that down-regulation of Cx43 expression in the U118 human glioma cell line increased migration by reducing cell-ECM adhesion, and changed the migration pattern from collective to single cell. In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail. Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration. Taken together our findings reveal an unexplored role of GJIC in facilitating collective migration.

Podoplanin: a marker for reactive gliosis in gliomas and brain injury.

Reactive astrogliosis is associated with many pathologic processes in the central nervous system, including gliomas. The glycoprotein podoplanin (PDPN) is upregulated in malignant gliomas. Using a syngeneic intracranial glioma mouse model, we show that PDPN is highly expressed in a subset of glial fibrillary acidic protein-positive astrocytes within and adjacent to gliomas. The expression of PDPN in tumor-associated reactive astrocytes was confirmed by its colocalization with the astrocytic marker S100ß and with connexin43, a major astrocytic gap junction protein. To determine whether the increase in PDPN is a general feature of gliosis, we used 2 mouse models in which astrogliosis was induced either by a needle injury or ischemia and observed similar upregulation of PDPN in reactive astrocytes in both models. Astrocytic PDPN was also found to be coexpressed with nestin, an intermediate filament marker for neural stem/progenitor cells. Our findings confirm that expression of PDPN is part of the normal host response to brain injury and gliomas, and suggest that it may be a novel cell surface marker for a specific population of reactive astrocytes in the vicinity of gliomas and nonneoplastic brain lesions. The findings also highlight the heterogeneity of glial fibrillary acidic protein-positive astrocytes in reactive gliosis.

Pannexin 2 protein expression is not restricted to the CNS.

Pannexins (Panx) are proteins homologous to the invertebrate gap junction proteins called innexins (Inx) and are traditionally described as transmembrane channels connecting the intracellular and extracellular compartments. Three distinct Panx paralogs (Panx1, Panx2 and Panx3) have been identified in vertebrates but previous reports on Panx expression and functionality focused primarily on Panx1 and Panx3 proteins. Several gene expression studies reported that Panx2 transcript is largely restricted to the central nervous system (CNS) hence suggesting that Panx2 might serve an important role in the CNS. However, the lack of suitable antibodies prevented the creation of a comprehensive map of Panx2 protein expression and Panx2 protein localization profile is currently mostly inferred from the distribution of its transcript. In this study, we characterized novel commercial monoclonal antibodies and surveyed Panx2 expression and distribution at the mRNA and protein level by real-time qPCR, Western blotting and immunofluorescence. Panx2 protein levels were readily detected in every tissue examined, even when transcriptional analysis predicted very low Panx2 protein expression. Furthermore, our results indicate that Panx2 transcriptional activity is a poor predictor of Panx2 protein abundance and does not correlate with Panx2 protein levels. Despite showing disproportionately high transcript levels, the CNS expressed less Panx2 protein than any other tissues analyzed. Additionally, we showed that Panx2 protein does not localize at the plasma membrane like other gap junction proteins but remains confined within cytoplasmic compartments. Overall, our results demonstrate that the endogenous expression of Panx2 protein is not restricted to the CNS and is more ubiquitous than initially predicted.

Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress.

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare autosomal dominant disease that results in visible developmental anomalies of the limbs, face, eyes and teeth. Recently analysis of human connexin43 (Cx43) DNA sequences has revealed a number of different missense, duplication and frame shift mutations resulting in this phenotype. A mouse model of this disorder has been created with a missense point mutation of the glycine amino acid at position 60 to serine (G60S). Heterozygote +/G60S mice exhibit a similar ODDD phenotype as observed in humans. In addition to the malformations listed above, ODDD patients often have neurological findings. In the brain, Cx43 is highly expressed in astrocytes and has been shown to play a role in neuroprotection. We were interested in determining the effect of the +/G60S mutation following stroke. Four days after middle cerebral artery occlusion the volume of infarct was larger in mice with the +/G60S mutation. In astrocyte-neuron co-cultures, exposure to glutamate also resulted in greater cellular death in the +/G60S mutants. Protein levels of Cx43 in the mutant mouse were found to be reduced when compared to the normal tissue. Cx43 protein was observed as a continual line of small punctate aggregates in the plasma membrane with increased intracellular localization, which is distinct from the larger plaques seen in the normal mouse astrocytes. Functionally, primary +/G60S astrocytes exhibited reduced gap junctional coupling and increased hemichannel activity, which may underlie the mechanism of increased damage during stroke. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.