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The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

Background: The endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux., Methods: We investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI)., Results: We show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells., Conclusions and General Significance: Our findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Francisco Rafael Martins Laurindo reports financial support was provided by State of Sao Paulo Research Foundation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background and Aims: Redox signaling is involved in the pathophysiology of aortic aneurysm/dissection. Protein Disulfide Isomerases and its prototype PDIA1 are thiol redox chaperones mainly from endoplasmic reticulum (ER), while PDIA1 cell surface pool redox-regulates thrombosis, cytoskeleton remodeling and integrin activation, which are mechanisms involved in aortic disease. Here we investigate the roles of PDIA1 in aortic dissection., Methods: Initially, we assessed the outcome of aortic aneurysm/dissection in transgenic PDIA1-overexpressing FVB mice using a model of 28-day exposure to lysyl oxidase inhibitor BAPN plus angiotensin-II infusion. In a second protocol, we assessed the effects of PDIA1 inhibitor isoquercetin (IQ) against aortic dissection in C57BL/6 mice exposed to BAPN for 28 days., Results: Transgenic PDIA1 overexpression associated with ca. 50% (p = 0.022) decrease (vs.wild-type) in mortality due to abdominal aortic rupture and protected against elastic fiber breaks in thoracic aorta. Conversely, exposure of mice to IQ increased thoracic aorta dissection-related mortality rates, from ca. 18%-50% within 28-days (p = 0.019); elastic fiber disruption and collagen deposition were also enhanced. The structurally-related compound diosmetin, which does not inhibit PDI, had negligible effects. In parallel, stretch-tension curves indicated that IQ amplified a ductile-type of biomechanical failure vs. control or BAPN-exposed mice aortas. IQ-induced effects seemed unassociated with nonspecific antioxidant effects or ER stress. In both models, echocardiographic analysis of surviving mice suggested that aortic rupture was dissociated from progressive dilatation., Conclusions: Our data indicate a protective role of PDIA1 against aortic dissection/rupture and potentially uncovers a novel integrative mechanism coupling redox and biomechanical homeostasis in vascular remodeling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆ lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Protein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1 G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3β and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.

Aim: Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice., Methods: Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed in vivo with magnetic resonance imaging and ex vivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO 2 - and NO 3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated., Results: The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine in vivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated ex vivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity., Conclusion: In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Background: Diabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it's role in HG-induced TECs remains to be elucidated. Methods: The HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB. Results: HG induced an increase in the expression of P4HB and TGFβ, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. Conclusion: P4HB regulated the TGFβ/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells. [ABSTRACT FROM AUTHOR]

Introduction: Diffuse pleural mesothelioma (DPM) of the pleura is a highly aggressive and treatment-resistant cancer linked to asbestos exposure. Despite multimodal treatment, the prognosis for DPM patients remains very poor, with an average survival of 2 years from diagnosis. Cisplatin, a platinum-based chemotherapy drug, is commonly used in the treatment of DPM. However, the development of resistance to cisplatin significantly limits its effectiveness, highlighting the urgent need for alternative therapeutic strategies. New selective inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) have shown promise in various malignancies by inhibiting cell cycle progression and suppressing tumor growth. Recent studies have indicated the potential of abemaciclib for DPM therapy, and a phase II clinical trial has shown preliminary encouraging results. Methods: Here, we tested abemaciclib, palbociclib, and ribociclib on a panel of DPM cell lines and non-tumor mesothelial(MET-5A) cells. Results: Specifically, we focused on abemaciclib, which was the mosteffective cytotoxic agent on all the DPM cell lines tested. Abemaciclib reduced DPM cell viability, clonogenic potential, and ability to grow as three-dimensional (3D) spheroids. In addition, abemaciclib induced prolonged effects, thereby impairing second-generation sphere formation and inducing G0/G1 arrest and apoptosis/necrosis. Interestingly, single silencing of RB family members did not impair cell response to abemaciclib, suggesting that they likely complement each other in triggering abemaciclib's cytostatic effect. Interestingly, abemaciclib reduced the phosphorylation of AKT, which is hyperactive in DPM and synergized with the pharmacological AKT inhibitor (AKTi VIII). Abemaciclib also synergized with cisplatin and reduced the viability of DPM cells with acquired resistance to cisplatin. Discussion: Overall, our results suggest that CDK4/6 inhibitors alone or in combination with standard of care should be further explored for DPM therapy. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) is the most frequently overexpressed receptor histologically exhibited by oral squamous cell carcinoma (OSCC) patients. Aberrated EGFR signaling may lead to recurrence and metastasis, thus laying the foundation of targeted therapy. Deactivating EGFR is likely to prevent downstream signaling thus resulting in apoptosis. Tyrosine kinase inhibitors (TKIs) have come into play to revert aggressiveness of OSCC. We exploited comparative proteomic analyses based on anti‐EGFR potential of varlitinib, using cellular proteomes from treated and untreated groups of oral cancer cells to identify protein players functional during oral carcinogenesis. Following separation by two-dimensional electrophoresis, differentially expressed cellular proteins (varlitinib-treated and untreated cells) were analyzed and later identified using QTOF mass spectrometer. In silico analysis for protein–protein interaction was carried out using STRING. Six differentially expressed proteins were identified as binding immunoglobulin protein (BiP), heat shock protein 7 C (HSP7C), protein disulfide isomerase 1 A (PDIA1), vimentin (VIME), keratin type I cytoskeletal 14 (K1C14), and β-Actin (ACTB). Relative expression of five proteins was found to be downregulated upon varlitinib treatment, whereas only K1C14 was upregulated in treated cells compared to control. Protein network analysis depicts the interaction between BiP, PDIA1, VIME, etc. indicating their role in oral carcinogenesis. Oral cancer cells show proteome shift based on varlitinib treatment compared to corresponding controls. Our data suggest candidature of varlitinib as a potent therapeutic agent and BiP, PDIA1, HSP7C, VIME, and β-Actin as complementary/prognostic markers of OSCC. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GBM) is a common malignant brain tumor with poor prognosis and high mortality. Numerous reports have identified the correlation between aging and the prognosis of patients with GBM. The purpose of this study was to establish a prognostic model for GBM patients based on aging-related gene (ARG) to help determine the prognosis of GBM patients. Methods: 143 patients with GBM from The Cancer Genomic Atlas (TCGA), 218 patients with GBM from the Chinese Glioma Genomic Atlas (CGGA) of China and 50 patients from Gene Expression Omnibus (GEO) were included in the study. R software (V4.2.1) and bioinformatics statistical methods were used to develop prognostic models and study immune infiltration and mutation characteristics. Results: Thirteen genes were screened out and used to establish the prognostic model finally, and the risk scores of the prognostic model was an independent factor (P < 0.001), which indicated a good prediction ability. In addition, there are significant differences in immune infiltration and mutation characteristics between the two groups with high and low risk scores. Conclusion: The prognostic model of GBM patients based on ARGs can predict the prognosis of GBM patients. However, this signature requires further investigation and validation in larger cohort studies. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is an important organelle that controls the intracellular and extracellular environments. The ER is responsible for folding almost one-third of the total protein population in the eukaryotic cell. Disruption of ER-protein folding is associated with numerous human diseases, including metabolic disorders, neurodegenerative diseases, and cancer. During ER perturbations, the cells deploy various mechanisms to increase the ER-folding capacity and reduce ER-protein load by minimizing the number of substrates entering the ER to regain homeostasis. These mechanisms include signaling pathways, degradation mechanisms, and other processes that mediate the reflux of ER content to the cytosol. In this review, we will discuss the recent discoveries of five different ER quality control mechanisms, including the unfolded protein response (UPR), ER-associated-degradation (ERAD), pre-emptive quality control, ER-phagy and ER to cytosol signaling (ERCYS). We will discuss the roles of these processes in decreasing ER-protein load and inter-mechanism crosstalk., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Mesenchymal stem cell-based acellular therapies have been widely exploited in managing hypertrophic scars. However, low maintenance dose and transitory therapeutic effects during topical medication remain a thorny issue. Herein, this study aimed to optimize the curative effect of adipose-derived stem cell conditioned medium (ADSC-CM) in the prevention of hypertrophic scarring. Methods: In the present study, ADSC-CM was concentrated via the freeze-drying procedure. The efficacy of different dose groups (CM, CM5, CM10) was conducted on the proliferation, apoptosis, and α-smooth muscle actin (α-SMA) expression of human keloid fibroblasts (HKFs) in vitro. Incorporation of adipose-derived stem cell concentrated conditioned medium (ADSCC-CM) into polysaccharide hydrogel was investigated in rabbit ear, in vivo. Haematoxylin-eosin (H&E) and Masson's trichrome staining were performed for the evaluation of scar hyperplasia. Results: We noted that ADSCC-CM could downregulate the α-SMA expression of HKFs in a dose-dependent manner. In the rabbit ear model, the scar hyperplasia in the medium-dose group (CM5) and high-dose group (CM10) was inhibited with reduced scar elevation index (SEI) under 4 months of observation. It is noteworthy that the union of CM5 and polysaccharide hydrogel (CM5+H) yielded the best preventive effect on scar hyperplasia. Briefly, melanin, height, vascularity, and pliability in the CM5+H group were better than those of the control group. Collagen was evenly distributed, and skin appendages could be regenerated. Conclusions: Altogether, ADSCC-CM can downregulate the expression of α-SMA due to its anti-fibrosis effect and promote the rearrangement of collagen fibres, which is integral to scar precaution. The in situ cross bonding of ADSCC-CM and polysaccharide hydrogel could remarkably enhance the therapeutic outcomes in inhibiting scar proliferation. Hence, the alliance of ADSCC-CM and hydrogel may become a potential alternative in hypertrophic scar prophylaxis. [ABSTRACT FROM AUTHOR]

Castration-resistant prostate cancer (CRPC) is a progressed stage of prostate cancer, which requires better understanding of the mechanisms and remains an unmet clinical need. As a common oncogene, K-Ras is associated with malignant behavior in different types of tumors but its role in CRPC is unknown. The present study aims to find the mechanism of K-Ras in CRPC and whether it can be used as a crucial molecule for the treatment of CRPC. For this purpose, tissue samples from primary prostate cancer (PPC) and CRPC patients were analyzed by immunohistochemistry and the data showed that K-Ras was elevated in CRPC. More importantly, higher K-Ras expression was related to a shorter recurrence-free survival time in patients with CRPC. In addition, K-Ras promoted the invasion, migration, and drug resistance of CRPC cells by activation of PLCε/PKCε signaling pathway. Meanwhile, the inhibitor of K-RasG12C mutants was able to inhibit malignant behavior of CRPC cells in vitro and in vivo. Inhibitors of K-RasG12C mutants have entered clinical trials. Taken together, the study shows that K-Ras may activate PKCε through PLCε, resulting in the alterations of malignant behavior of CRPC. Inhibitor 9, an inhibitor of the K-RasG12C mutant, has a strong anti-tumor effect in CRPC, which potentially suggests that inhibitors of this nature may serve as a promising treatment for CRPC. [ABSTRACT FROM AUTHOR]

Since the discovery of the proto-oncogene Wnt1 (Int1) in 1982, WNT signaling has been identified as one of the most important pathways that regulates a wide range of fundamental developmental and physiological processes in multicellular organisms. The canonical WNT signaling pathway depends on the stabilization and translocation of β-catenin and plays important roles in development and homeostasis. The WNT/planar cell polarity (WNT/PCP) signaling, also known as one of the β-catenin-independent WNT pathways, conveys directional information to coordinate polarized cell behaviors. Similar to WNT/β-catenin signaling, disruption or aberrant activation of WNT/PCP signaling also underlies a variety of developmental defects and cancers. However, the pharmacological targeting of WNT/PCP signaling for therapeutic purposes remains largely unexplored. In this review, we briefly discuss WNT/PCP signaling in development and disease and summarize the known drugs/inhibitors targeting this pathway., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)