Programmable self-regulated molecular buffers for precise sustained drug delivery.

Unlike artificial nanosystems, biological systems are ideally engineered to respond to their environment. As such, natural molecular buffers ensure precise and quantitative delivery of specific molecules through self-regulated mechanisms based on Le Chatelier's principle. Here, we apply this principle to design self-regulated nucleic acid molecular buffers for the chemotherapeutic drug doxorubicin and the antimalarial agent quinine. We show that these aptamer-based buffers can be programmed to maintain any specific desired concentration of free drug both in vitro and in vivo and enable the optimization of the chemical stability, partition coefficient, pharmacokinetics and biodistribution of the drug. These programmable buffers can be built from any polymer and should improve patient therapeutic outcome by enhancing drug activity and minimizing adverse effects and dosage frequency.

A Degradation Motif in STAU1 Defines a Novel Family of Proteins Involved in Inflammation.

Cancer development is regulated by inflammation. Staufen1 (STAU1) is an RNA-binding protein whose expression level is critical in cancer cells as it is related to cell proliferation or cell death. STAU1 protein levels are downregulated during mitosis due to its degradation by the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). In this paper, we map the molecular determinant involved in STAU1 degradation to amino acids 38-50, and by alanine scanning, we shorten the motif to F39PxPxxLxxxxL50 (FPL-motif). Mutation of the FPL-motif prevents STAU1 degradation by APC/C. Interestingly, a search in databases reveals that the FPL-motif is shared by 15 additional proteins, most of them being involved in inflammation. We show that one of these proteins, MAP4K1, is indeed degraded via the FPL-motif; however, it is not a target of APC/C. Using proximity labeling with STAU1, we identify TRIM25, an E3 ubiquitin ligase involved in the innate immune response and interferon production, as responsible for STAU1 and MAP4K1 degradation, dependent on the FPL-motif. These results are consistent with previous studies that linked STAU1 to cancer-induced inflammation and identified a novel degradation motif that likely coordinates a novel family of proteins involved in inflammation. Data are available via ProteomeXchange with the identifier PXD036675.

Phosphomimicry on STAU1 Serine 20 Impairs STAU1 Posttranscriptional Functions and Induces Apoptosis in Human Transformed Cells.

Staufen 1 (STAU1) is an RNA-binding protein that is essential in untransformed cells. In cancer cells, it is rather STAU1 overexpression that impairs cell proliferation. In this paper, we show that a modest increase in STAU1 expression in cancer cells triggers apoptosis as early as 12 h post-transfection and impairs proliferation in non-apoptotic cells for several days. Interestingly, a mutation that mimics the phosphorylation of STAU1 serine 20 is sufficient to cause these phenotypes, indicating that serine 20 is at the heart of the molecular mechanism leading to apoptosis. Mechanistically, phosphomimicry on serine 20 alters the ability of STAU1 to regulate translation and the decay of STAU1-bound mRNAs, indicating that the posttranscriptional regulation of mRNAs by STAU1 controls the balance between proliferation and apoptosis. Unexpectedly, the expression of RBD2S20D, the N-terminal 88 amino acids with no RNA-binding activity, is sufficient to induce apoptosis via alteration, in trans, of the posttranscriptional functions of endogenous STAU1. These results suggest that STAU1 is a sensor that controls the balance between cell proliferation and apoptosis, and, therefore, may be considered as a novel therapeutic target against cancer.

The double-stranded RNA-binding protein, Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation.

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5'untranslated region (5'UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.

The Staufen1-dependent cell cycle regulon or how a misregulated RNA-binding protein leads to cancer.

In recent years, an increasing number of reports have linked the RNA-binding protein Staufen1 (STAU1) to the control of cell decision making. In non-transformed cells, STAU1 balances the expression of messenger RNA (mRNA) regulons that regulate differentiation and well-ordered cell division. Misregulation of STAU1 expression and/or functions changes the fragile balance in the expression of pro- and anti-proliferative and apoptotic genes and favours a novel equilibrium that supports cell proliferation and cancer development. The misregulation of STAU1 functions causes multiple coordinated modest effects in the post-transcriptional regulation of many RNA targets that code for cell cycle regulators, leading to dramatic consequences at the cellular level. The new tumorigenic equilibrium in STAU1-mediated gene regulation observed in cancer cells can be further altered by a slight increase in STAU1 expression that favours expression of pro-apoptotic genes and cell death. The STAU1-dependent cell cycle regulon is a good model to study how abnormal expression of an RNA-binding protein promotes cell growth and provides an advantageous selection of malignant cells in the first step of cancer development.

STAU2 protein level is controlled by caspases and the CHK1 pathway and regulates cell cycle progression in the non-transformed hTERT-RPE1 cells.

BACKGROUND: Staufen2 (STAU2) is an RNA binding protein involved in the posttranscriptional regulation of gene expression. In neurons, STAU2 is required to maintain the balance between differentiation and proliferation of neural stem cells through asymmetric cell division. However, the importance of controlling STAU2 expression for cell cycle progression is not clear in non-neuronal dividing cells. We recently showed that STAU2 transcription is inhibited in response to DNA-damage due to E2F1 displacement from the STAU2 gene promoter. We now study the regulation of STAU2 steady-state levels in unstressed cells and its consequence for cell proliferation. RESULTS: CRISPR/Cas9-mediated and RNAi-dependent STAU2 depletion in the non-transformed hTERT-RPE1 cells both facilitate cell proliferation suggesting that STAU2 expression influences pathway(s) linked to cell cycle controls. Such effects are not observed in the CRISPR STAU2-KO cancer HCT116 cells nor in the STAU2-RNAi-depleted HeLa cells. Interestingly, a physiological decrease in the steady-state level of STAU2 is controlled by caspases. This effect of peptidases is counterbalanced by the activity of the CHK1 pathway suggesting that STAU2 partial degradation/stabilization fines tune cell cycle progression in unstressed cells. A large-scale proteomic analysis using STAU2/biotinylase fusion protein identifies known STAU2 interactors involved in RNA translation, localization, splicing, or decay confirming the role of STAU2 in the posttranscriptional regulation of gene expression. In addition, several proteins found in the nucleolus, including proteins of the ribosome biogenesis pathway and of the DNA damage response, are found in close proximity to STAU2. Strikingly, many of these proteins are linked to the kinase CHK1 pathway, reinforcing the link between STAU2 functions and the CHK1 pathway. Indeed, inhibition of the CHK1 pathway for 4 h dissociates STAU2 from proteins involved in translation and RNA metabolism. CONCLUSIONS: These results indicate that STAU2 is involved in pathway(s) that control(s) cell proliferation, likely via mechanisms of posttranscriptional regulation, ribonucleoprotein complex assembly, genome integrity and/or checkpoint controls. The mechanism by which STAU2 regulates cell growth likely involves caspases and the kinase CHK1 pathway.

High Level of Staufen1 Expression Confers Longer Recurrence Free Survival to Non-Small Cell Lung Cancer Patients by Promoting THBS1 mRNA Degradation.

Stau1 is a pluripotent RNA-binding protein that is responsible for the post-transcriptional regulation of a multitude of transcripts. Here, we observed that lung cancer patients with a high Stau1 expression have a longer recurrence free survival. Strikingly, Stau1 did not impair cell proliferation in vitro, but rather cell migration and cell adhesion. In vivo, Stau1 depletion favored tumor progression and metastases development. In addition, Stau1 depletion strongly impaired vessel maturation. Among a panel of candidate genes, we specifically identified the mRNA encoding the cell adhesion molecule Thrombospondin 1 (THBS1) as a new target for Staufen-mediated mRNA decay. Altogether, our results suggest that regulation of THBS1 expression by Stau1 may be a key process involved in lung cancer progression.

Staufen1 localizes to the mitotic spindle and controls the localization of RNA populations to the spindle.

Staufen1 (STAU1) is an RNA-binding protein involved in the post-transcriptional regulation of mRNAs. We report that a large fraction of STAU1 localizes to the mitotic spindle in colorectal cancer HCT116 cells and in non-transformed hTERT-RPE1 cells. Spindle-associated STAU1 partly co-localizes with ribosomes and active sites of translation. We mapped the molecular determinant required for STAU1-spindle association within the first 88 N-terminal amino acids, a domain that is not required for RNA binding. Interestingly, transcriptomic analysis of purified mitotic spindles revealed that 1054 mRNAs and the precursor ribosomal RNA (pre-rRNA), as well as the long non-coding RNAs and small nucleolar RNAs involved in ribonucleoprotein assembly and processing, are enriched on spindles compared with cell extracts. STAU1 knockout causes displacement of the pre-rRNA and of 154 mRNAs coding for proteins involved in actin cytoskeleton organization and cell growth, highlighting a role for STAU1 in mRNA trafficking to spindle. These data demonstrate that STAU1 controls the localization of subpopulations of RNAs during mitosis and suggests a novel role of STAU1 in pre-rRNA maintenance during mitosis, ribogenesis and/or nucleoli reassembly.

Staufen1 is Essential for Cell-Cycle Transitions and Cell Proliferation Via the Control of E2F1 Expression.

Cell cycle is a highly regulated process that is finely coordinated by a plethora of interconnected regulators. In this paper, we report that post-transcriptional mechanisms mediated by the RNA-binding protein Staufen1 (STAU1) are essential for the proliferation of non-transformed cells (hTERT-RPE1 and IMR90). Cell sorting quantification and time-lapse video microscopy using FUCCI-hTERT-RPE1 cells identified the G1/S and G2/M phase transitions of the cell cycle as crucial steps for STAU1 functions. The level of expression of 35 transcripts coding for cell-cycle regulators is up- or down-regulated following STAU1 depletion. Among others, expression of E2F1, a transcription factor essential for the G1/S transition, is decreased in STAU1 depleted cells, dependent on a STAU1-binding site in the 3' untranslated region of E2F1 mRNA. Interestingly, E2F1, in turn, increases STAU1 transcription, highlighting a regulatory loop that enhances expression of both STAU1 and E2F1. Our results indicate that a STAU1-mediatedpost-transcriptional mechanism of gene regulation controls an mRNA regulon involved in decision making during cell-cycle phase transitions and that this mechanism is essential for cell-cycle progression in non-tumor cells.

HIV-1 requires Staufen1 to dissociate stress granules and to produce infectious viral particles.

The human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) has two major fates during viral replication: to serve as the template for the major structural and enzymatic proteins, or to be encapsidated and packaged into assembling virions to serve as the genomic vRNA in budding viruses. The dynamic balance between vRNA translation and encapsidation is mediated by numerous host proteins, including Staufen1. During HIV-1 infection, HIV-1 recruits Staufen1 to assemble a distinct ribonucleoprotein complex promoting vRNA encapsidation and viral assembly. Staufen1 also rescues vRNA translation and gene expression during conditions of cellular stress. In this work, we utilized novel Staufen1-/- gene-edited cells to further characterize the contribution of Staufen1 in HIV-1 replication. We observed a marked deficiency in the ability of HIV-1 to dissociate stress granules (SGs) in Staufen1-deficient cells and remarkably, the vRNA repositioned to SGs. These phenotypes were rescued by Staufen1 expression in trans or in cis, but not by a dsRBD-binding mutant, Staufen1F135A. The mistrafficking of the vRNA in these Staufen1-/- cells was also accompanied by a dramatic decrease in viral production and infectivity. This work provides novel insight into the mechanisms by which HIV-1 uses Staufen1 to ensure optimal vRNA translation and trafficking, supporting an integral role for Staufen1 in the HIV-1 life cycle, positioning it as an attractive target for next-generation antiretroviral agents.

Correction to: The M-phase specific hyperphosphorylation of Staufen2 involved the cyclin-dependent kinase CDK1.

Following publication of the original article [1], the authors reported a change to one of the author names.

The M-phase specific hyperphosphorylation of Staufen2 involved the cyclin-dependent kinase CDK1.

BACKGROUND: Staufen2 (STAU2) is an RNA-binding protein involved in the post-transcriptional regulation of gene expression. This protein was shown to be required for organ formation and cell differentiation. Although STAU2 functions have been reported in neuronal cells, its role in dividing cells remains deeply uncharacterized. Especially, its regulation during the cell cycle is completely unknown. RESULTS: In this study, we showed that STAU2 isoforms display a mitosis-specific slow migration pattern on SDS-gels in all tested transformed and untransformed cell lines. Deeper analyses in hTert-RPE1 and HeLa cells further indicated that the slow migration pattern of STAU2 isoforms is due to phosphorylation. Time course studies showed that STAU2 phosphorylation occurs before prometaphase and terminates as cells exit mitosis. Interestingly, STAU2 isoforms were phosphorylated on several amino acid residues in the C-terminal half via the cyclin-dependent kinase 1 (Cdk1), an enzyme known to play crucial roles during mitosis. Introduction of phospho-mimetic or phospho-null mutations in STAU2 did not impair its RNA-binding capacity, its stability, its interaction with protein co-factors or its sub-cellular localization, suggesting that STAU2 phosphorylation in mitosis does not regulate these functions. Similarly, STAU2 phosphorylation is not likely to be crucial for cell cycle progression since expression of phosphorylation mutants in hTert-RPE1 cells did not impair cell proliferation. CONCLUSIONS: Altogether, these results indicate that STAU2 isoforms are phosphorylated during mitosis and that the phosphorylation process involves Cdk1. The meaning of this post-translational modification is still elusive.

The downregulation of the RNA-binding protein Staufen2 in response to DNA damage promotes apoptosis.

Staufen2 (Stau2) is an RNA-binding protein involved in cell fate decision by controlling several facets of mRNA processing including localization, splicing, translation and stability. Herein we report that exposure to DNA-damaging agents that generate replicative stress such as camptothecin (CPT), 5-fluoro-uracil (5FU) and ultraviolet radiation (UVC) causes downregulation of Stau2 in HCT116 colorectal cancer cells. In contrast, other agents such as doxorubicin and ionizing radiation had no effect on Stau2 expression. Consistently, Stau2 expression is regulated by the ataxia telangiectasia and Rad3-related (ATR) signaling pathway but not by the DNA-PK or ataxia telangiectasia mutated/checkpoint kinase 2 pathways. Stau2 downregulation is initiated at the level of transcription, independently of apoptosis induction. Promoter analysis identified a short 198 bp region which is necessary and sufficient for both basal and CPT-regulated Stau2 expression. The E2F1 transcription factor regulates Stau2 in untreated cells, an effect that is abolished by CPT treatment due to E2F1 displacement from the promoter. Strikingly, Stau2 downregulation enhances levels of DNA damage and promotes apoptosis in CPT-treated cells. Taken together our results suggest that Stau2 is an anti-apoptotic protein that could be involved in DNA replication and/or maintenance of genome integrity and that its expression is regulated by E2F1 via the ATR signaling pathway.

Regional expression and ultrastructural localization of EphA7 in the hippocampus and cerebellum of adult rat.

EphA7 is expressed in the adult central nervous system (CNS), where its roles are yet poorly defined. We mapped its distribution using in situ hybridization (ISH) and immunohistochemistry (IHC) combined with light (LM) and electron microscopy (EM) in adult rat and mouse brain. The strongest ISH signal was in the hippocampal pyramidal and granule cell layers. Moderate levels were detected in habenula, striatum, amygdala, the cingulate, piriform and entorhinal cortex, and in cerebellum, notably the Purkinje cell layer. The IHC signal distribution was consistent with ISH results, with transport of the protein to processes, as exemplified in the hippocampal neuropil layers and weakly stained pyramidal cell layers. In contrast, in the cerebellum, the Purkinje cell bodies were the most strongly immunolabeled elements. EM localized the cell surface-expression of EphA7 essentially in postsynaptic densities (PSDs) of dendritic spines and shafts, and on some astrocytic leaflets, in both hippocampus and cerebellum. Perikaryal and dendritic labeling was mostly intracellular, associated with the synthetic and trafficking machineries. Immunopositive vesicles were also observed in axons and axon terminals. Quantitative analysis in EM showed significant differences in the frequency of labeled elements between regions. Notably, labeled dendrites were ∼3-5 times less frequent in cerebellum than in hippocampus, but they were individually endowed with ∼10-40 times higher frequencies of PSDs, on their shafts and spines. The cell surface localization of EphA7, being preferentially in PSDs, and in perisynaptic astrocytic leaflets, provides morphologic evidence that EphA7 plays key roles in adult CNS synaptic maintenance, plasticity, or function. J. Comp. Neurol. 524:2462-2478, 2016. © 2016 Wiley Periodicals, Inc.

Cell cycle-dependent regulation of the RNA-binding protein Staufen1.

Staufen1 (Stau1) is a ribonucleic acid (RNA)-binding protein involved in the post-transcriptional regulation of gene expression. Recent studies indicate that Stau1-bound messenger RNAs (mRNAs) mainly code for proteins involved in transcription and cell cycle control. Consistently, we report here that Stau1 abundance fluctuates through the cell cycle in HCT116 and U2OS cells: it is high from the S phase to the onset of mitosis and rapidly decreases as cells transit through mitosis. Stau1 down-regulation is mediated by the ubiquitin-proteasome system and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Stau1 interacts with the APC/C co-activators Cdh1 and Cdc20 via its first 88 N-terminal amino acids. The importance of controlling Stau155 levels is underscored by the observation that its overexpression affects mitosis entry and impairs proliferation of transformed cells. Microarray analyses identified 275 Stau1(55)-bound mRNAs in prometaphase cells, an early mitotic step that just precedes Stau1 degradation. Interestingly, several of these mRNAs are more abundant in Stau155-containing complexes in cells arrested in prometaphase than in asynchronous cells. Our results point out for the first time to the possibility that Stau1 participates in a mechanism of post-transcriptional regulation of gene expression that is linked to cell cycle progression in cancer cells.

Dopamine facilitates dendritic spine formation by cultured striatal medium spiny neurons through both D1 and D2 dopamine receptors.

Variations of dopamine (DA) levels induced by drugs of abuse or in the context of Parkinson's disease modulate the number of dendritic spines in medium spiny neurons (MSNs) of the striatum, showing that DA plays a major role in the structural plasticity of MSNs. However, little is presently known regarding early spine development in MSNs occurring before the arrival of cortical inputs and in particular about the role of DA and D1 (D1R) and D2 (D2R) DA receptors. A cell culture model reconstituting early cellular interactions between MSNs, intrinsic cholinergic interneurons and DA neurons was used to study the role of DA in spine formation. After 5 or 10 days in vitro, the presence of DA neurons increased the number of immature spine-like protrusions. In MSN monocultures, chronic activation of D1R or D2R also increased the number of spines and spinophilin expression in MSNs, suggesting a direct role for these receptors. In DA-MSN cocultures, chronic blockade of D1R or D2R reduced the number of dendritic spines. Interestingly, the combined activation or blockade of both D1R and D2R failed to elicit more extensive spine formation, suggesting that both receptors act through a mechanism that is not additive. Finally, we found increased ionotropic glutamate receptor responsiveness and miniature excitatory postsynaptic current (EPSC) frequency in DA-MSN co-cultures, in parallel with a higher number of spines containing PSD-95, suggesting that the newly formed spines present functional post-synaptic machinery preparing the MSNs to receive additional glutamatergic contacts. These results represent a first step in the understanding of how dopamine neurons promote the structural plasticity of MSNs during the development of basal ganglia circuits.

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing.

In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.

PTHrP(1-34)-mediated repression of the PHEX gene in osteoblastic cells involves the transcriptional repressor E4BP4.

PHosphate-regulating gene with homology to Endopeptidase on the X chromosome (PHEX) has been identified as the gene mutated in X-linked hypophosphatemia (XLH) syndrome, the most prevalent form of rickets in humans. The predominant expression of PHEX in bones and teeth, and the defective mineralization of these tissues in XLH patients indicate that PHEX is an important regulator of mineralization. Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are known to regulate the expression of numerous genes in osteoblastic cells through activation of the protein kinase A pathway, including repression of PHEX. PTH also activates the transcriptional repressor E4BP4 through the same pathway, suggesting that PTH or PTHrP-mediated repression of PHEX expression could involve E4BP4. To evaluate this possibility, we treated UMR-106 osteoblastic cells with PTHrP(1-34), and used RT-PCR and immunoblotting to analyze PHEX and E4BP4 expression. E4BP4 mRNA and protein levels were rapidly increased in cells treated with PTHrP(1-34), with a concomitant decrease in PHEX expression. This downregulation of PHEX could be reproduced by overexpression of E4BP4. Moreover, PTHrP(1-34)-mediated PHEX repression was blocked when cells were transfected with a siRNA targeting E4BP4 mRNA. Finally, DNA pull-down and luciferase assays showed that two E4BP4 response elements located in PHEX promoter were functional. These results underline the important role of E4BP4 in osteoblastic cells and further define the repression mechanism of PHEX gene by PTHrP(1-34).

Fragile X related protein 1 clusters with ribosomes and messenger RNAs at a subset of dendritic spines in the mouse hippocampus.

The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.