METTL3‐m6 A methylase regulates the osteogenic potential of bone marrow mesenchymal stem cells in osteoporotic rats via the Wnt signalling pathway

ObjectivesBone marrow mesenchymal stem cells (BMSCs) hold a high osteogenic differentiation potential, but the mechanisms that control the osteogenic ability of BMSCs from osteoporosis (OP‐BMSCs) need further research. The purpose of this experiment is to discuss the osteogenic effect of Mettl3 on OP‐BMSCs and explore new therapeutic target that can enhance the bone formation ability of OP‐BMSCs.Materials and MethodsThe bilateral ovariectomy (OVX) method was used to establish the SD rat OP model. Dot blots were used to reveal the different methylation levels of BMSCs and OP‐BMSCs. Lentiviral‐mediated overexpression of Mettl3 was applied in OP‐BMSCs. QPCR and WB detected the molecular changes of osteogenic‐related factors and Wnt signalling pathway in vitro experiment. The staining of calcium nodules and alkaline phosphatase detected the osteogenic ability of OP‐BMSCs. Micro‐CT and histological examination evaluated the osteogenesis of Mettl3 in OP rats in vivo.ResultsThe OP rat model was successfully established by OVX. Methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro experiment, overexpression of Mettl3 could upregulate the osteogenic‐related factors and activate the Wnt signalling pathway in OP‐BMSCs. However, osteogenesis of OP‐BMSCs was weakened by treatment with the canonical Wnt inhibitor Dickkopf‐1. Micro‐CT showed that the Mettl3(+) group had an increased amount of new bone formation at 8 weeks. Moreover, the results of histological staining were the same as the micro‐CT results.ConclusionsTaken together, the methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro and in vivo studies, overexpression of Mettl3 could partially rescue the decreased bone formation ability of OP‐BMSCs by the canonical Wnt signalling pathway. Therefore, Mettl3 may be a key targeted gene for bone generation and therapy of bone defects in OP patients.

In this study, the osteoporosis rat model was successfully established by OVX. OP‐BMSCs were successfully isolated and cultured from the femur of OP rat. Lentiviral‐mediated overexpression of Mettl3 could partially rescue the impaired osteogenic ability of OP‐BMSCs by activating the canonical Wnt signalling pathway in vitro and in vivo .     

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C

Mycobacterium tuberculosis virulence is highly metal‐dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu²⁺ ≥ Zn²⁺ > Ni²⁺ > Co²⁺) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k _cat rather than K _M. Removal of the artificial N‐terminal 6xHis‐tag did not change the metal‐dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn²⁺ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal‐dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.     

Plasmodium chitinases: revisiting a target of transmission‐blockade against malaria

Malaria is a life‐threatening disease caused by one of the five species of Plasmodium, among which Plasmodium falciparum cause the deadliest form of the disease. Plasmodium species are dependent on a vertebrate host and a blood‐sucking insect vector to complete their life cycle. Plasmodium chitinases belonging to the GH18 family are secreted inside the mosquito midgut, during the ookinete stage of the parasite. Chitinases mediate the penetration of parasite through the peritrophic membrane, facilitating access to the gut epithelial layer. In this review, we describe Plasmodium chitinases with special emphasis on chitinases from P. falciparum and P. vivax, the representative examples of the short and long forms of this protein. In addition to the chitinase domain, chitinases belonging to the long form contain a pro‐domain and chitin‐binding domain. Amino acid sequence alignment of long and short form chitinase domains reveals multiple positions containing variant residues. A subset of these positions was found to be conserved or invariant within long or short forms, indicating the role of these positions in attributing form‐specific activity. The reported differences in affinities to allosamidin for P. vivax and P. falciparum were predicted to be due to different residues at two amino acid positions, resulting in altered interactions with the inhibitor. Understanding the role of these amino acids in Plasmodium chitinases will help us elucidate the mechanism of catalysis and the mode of inhibition, which will be the key for identification of potent inhibitors or antibodies demonstrating transmission‐blocking activity.     

Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity

Upon Mycobacterium tuberculosis (Mtb) infection, protein kinase G (PknG), a eukaryotic‐type serine‐threonine protein kinase (STPK), is secreted into host macrophages to promote intracellular survival of the pathogen. However, the mechanisms underlying this PknG–host interaction remain unclear. Here, we demonstrate that PknG serves both as a ubiquitin‐activating enzyme (E1) and a ubiquitin ligase (E3) to trigger the ubiquitination and degradation of tumor necrosis factor receptor‐associated factor 2 (TRAF2) and TGF‐β‐activated kinase 1 (TAK1), thereby inhibiting the activation of NF‐κB signaling and host innate responses. PknG promotes the attachment of ubiquitin (Ub) to the ubiquitin‐conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82‐Ub), rather than the usual C86‐Ub thiol‐ester bond. PknG induces the discharge of Ub from UbcH7 by acting as an isopeptidase, before attaching Ub to its substrates. These results demonstrate that PknG acts as an unusual ubiquitinating enzyme to remove key components of the innate immunity system, thus providing a potential target for tuberculosis treatment.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

HLA Allele E*01:01 Is Associated with a Reduced Risk of EBV-Related Classical Hodgkin Lymphoma Independently of HLA-A*01/*02

Background

An inefficient immune response against Epstein-Barr virus (EBV) infection is related to the pathogenesis of a subgroup of classical Hodgkin lymphomas (cHL). Some EBV immune-evasion mechanisms target HLA presentation, including the non-classical HLA-E molecule. HLA-E can be recognized by T cells via the TCR, and it also regulates natural killer (NK) cell signaling through the inhibitory CD94/NKG2A receptor. Some evidences indicate that EBV-infected B-cells promote the proliferation of NK subsets bearing CD94/NKG2A, suggesting a relevant function of these cells in EBV control. Variations in CD94/NKG2A-HLA-E interactions could affect NK cell-mediated immunity and, consequently, play a role in EBV-driven transformation and lymphomagenesis. The two most common HLA-E alleles, E*01:01 and E*01:03, differ by a single amino acid change that modifies the molecule function. We hypothesized that the functional differences in these variants might participate in the pathogenicity of EBV.

Aim

We studied two series of cHL patients, both with EBV-positive and-negative cases, and a cohort of unrelated controls, to assess the impact of HLA-E variants on EBV-related cHL susceptibility.

Results

We found that the genotypes with at least one copy of E*01:01 (i.e., E*01:01 homozygous and heterozygous) were underrepresented among cHL patients from both series compared to controls (72.6% and 71.6% vs 83%, p = 0.001). After stratification by EBV status, we found low rates of E*01:01-carriers mainly among EBV-positive cases (67.6%). These reduced frequencies are seen independently of other factors such as age, gender, HLA-A*01 and HLA-A*02, HLA alleles positively and negatively associated with the disease (adjusted OR = 0.4, p = 0.001). Furthermore, alleles from both HLA loci exert a cumulative effect on EBV-associated cHL susceptibility.

Conclusions

These results indicate that E*01:01 is a novel protective genetic factor in EBV-associated cHL and support a role for HLA-E recognition on the control of EBV infection and lymphomagenesis.     

BCAS3 exhibits oncogenic properties by promoting CRL4A‐mediated ubiquitination of p53 in breast cancer

ObjectivesBreast cancer‐amplified sequence 3 (BCAS3) was initially found to be amplified in human breast cancer (BRCA); however, there has been little consensus on the functions of BCAS3 in breast tumours.Materials and methodsWe analysed BCAS3 expression in BRCA using bio‐information tools. Affinity purification and mass spectrometry were employed to identify BCAS3‐associated proteins. GST pull‐down and ubiquitination assays were performed to analyse the interaction mechanism between BCAS3/p53 and CUL4A‐RING E3 ubiquitin ligase (CRL4A) complex. BCAS3 was knocked down individually or in combination with p53 in MCF‐7 cells to further explore the biological functions of the BCAS3/p53 axis. The clinical values of BCAS3 for BRCA progression were evaluated via semiquantitative immunohistochemistry (IHC) analysis and Cox regression.ResultsWe reported that the expression level of BCAS3 in BRCA was higher than that in adjacent normal tissues. High BCAS3 expression promoted growth, inhibited apoptosis and conferred chemoresistance in breast cancer cells. Mechanistically, BCAS3 overexpression fostered BRCA cell growth by interacting with the CRL4A complex and promoting ubiquitination and proteasomal degradation of p53. Furthermore, BCAS3 could regulate cell growth, apoptosis and chemoresistance through a p53‐mediated mechanism. Clinically, BCAS3 overexpression was significantly correlated with a malignant phenotype. Moreover, higher expression of BCAS3 correlates with shorter overall survival (OS) in BRCA.ConclusionsThe functional characterization of BCAS3 offers new insights into the oncogenic properties and chemotherapy resistance in breast cancer.     

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4‐amino‐4,6‐dideoxy‐d‐glucose, also known as d‐viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide‐linked sugar, which in the Mimivirus is thought to be UDP‐d‐glucose. The enzyme required for the installment of the amino group at the C‐4′ position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5′‐phosphate‐dependent enzyme, referred to as L136. For this analysis, three high‐resolution X‐ray structures were determined: the wildtype enzyme/pyridoxamine 5′‐phosphate/dTDP complex and the site‐directed mutant variant K185A in the presence of either UDP‐4‐amino‐4,6‐dideoxy‐d‐glucose or dTDP‐4‐amino‐4,6‐dideoxy‐d‐glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP‐d‐glucose or dTDP‐d‐glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three‐dimensional architecture was previously reported by this laboratory. As determined in this investigation,DesI shows a profound preference in its catalytic efficiency for the dTDP‐linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three‐dimensional model for a virally encoded PLP‐dependent enzyme and thus provides new information on sugar aminotransferases in general.     

Study on the development and integration of 3D‐printed optics in small‐scale productions of single‐use cultivation vessels

Integrating optical sensors and 3D‐printed optics into single‐use (SU) cultivation vessels for customized, tailor‐made equipment could be a next big step in the bioreactor and screening platform development enabling online bioprocess monitoring. Many different parameters such as pH, oxygen, carbon dioxide and optical density (OD) can be monitored more easily using online measuring instruments compared to offline sampling. Space‐saving integrated sensors in combination with adapted optics such as prisms open up vastly new possibilities to precisely guide light through SU vessels. This study examines how optical prisms can be 3D‐printed with a 3D‐inkjet printer, modified and then evaluated in a custom made optical bench. The prisms are coated or bonded with thin cover glasses. For the examination of reflectance performance and conformity prisms are compared on the basis of measured characteristics of a conventional glass prism. In addition, the most efficient and reproducible prism geometry and modification technique is applied to a customized 3D‐printed cultivation vessel. The vessel is evaluated on a commercial sensor‐platform, a shake flask reader (SFR) vario, to investigate its sensing‐characteristics while monitoring scattered light with the turbidity standard formazine and a cell suspension of Saccharomyces cerevisiae as model organism. It is demonstrated that 3D‐printed prisms can be used in combination with commercial scattered light sensor‐platforms to determine OD of a microbial culture and that a 3D‐printed unibody design with integrated optics in a cultivation vessel is feasible. In the range of OD₆₀₀ 0–1.16 rel.AU a linear correlation between sensor amplitude and offline determined OD can be achieved. Thus, enabling for the first time a measurement of low cell densities with the SFR vario platform. Moreover, sensitivity is also at least three times higher compared to the commonly used method.     

Tnfaip2/exoc3‐driven lipid metabolism is essential for stem cell differentiation and organ homeostasis

Lipid metabolism influences stem cell maintenance and differentiation but genetic factors that control these processes remain to be delineated. Here, we identify Tnfaip2 as an inhibitor of reprogramming of mouse fibroblasts into induced pluripotent stem cells. Tnfaip2 knockout impairs differentiation of embryonic stem cells (ESCs), and knockdown of the planarian para‐ortholog, Smed‐exoc3, abrogates in vivo tissue homeostasis and regeneration—processes that are driven by somatic stem cells. When stimulated to differentiate, Tnfaip2‐deficient ESCs fail to induce synthesis of cellular triacylglycerol (TAG) and lipid droplets (LD) coinciding with reduced expression of vimentin (Vim)—a known inducer of LD formation. Smed‐exoc3 depletion also causes a strong reduction of TAGs in planarians. The study shows that Tnfaip2 acts epistatically with and upstream of Vim in impairing cellular reprogramming. Supplementing palmitic acid (PA) and palmitoyl‐L‐carnitine (the mobilized form of PA) restores the differentiation capacity of Tnfaip2‐deficient ESCs and organ maintenance in Smed‐exoc3‐depleted planarians. Together, these results identify a novel role of Tnfaip2 and exoc3 in controlling lipid metabolism, which is essential for ESC differentiation and planarian organ maintenance.     

An integrated approach reveals how lipo‐chitooligosaccharides interact with the lysin motif receptor‐like kinase MtLYR3

N‐acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant‐specific Lysin Motif Receptor‐Like Kinases (LysM‐RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo‐chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen‐fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM‐RLK LYR3 (MtLYR3) as a specific LCO‐binding protein. We also showed that the absence of LCO binding to LYR3 of the non‐mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site‐directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3‐LysM3 by those of MtLYR3‐LysM3 allowed the recovery of high‐affinity LCO binding in experimental radioligand‐binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.     

G6PD overexpression protects from oxidative stress and age‐related hearing loss

Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose‐6‐phosphate dehydrogenase (G6PD), which catalyzes the rate‐limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD‐Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild‐type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3‐month‐old G6PD‐Tg mice than in wild‐type counterparts, whereas the levels of pro‐apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL⁺ apoptotic cells were all lower in 9‐month‐old G6PD‐Tg than in wild‐type counterparts. Unexpectedly, G6PD overexpression triggered low‐grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.     

KCNJ10 May Not Be a Contributor to Nonsyndromic Enlargement of Vestibular Aqueduct (NSEVA) in Chinese Subjects

Background

Nonsyndromic enlargement of vestibular aqueduct (NSEVA) is an autosomal recessive hearing loss disorder that is associated with mutations in SLC26A4. However, not all patients with NSEVA carry biallelic mutations in SLC26A4. A recent study proposed that single mutations in both SLC26A4 and KCNJ10 lead to digenic NSEVA. We examined whether KCNJ10 excert a role in the pathogenesis of NSEVA in Chinese patients.

Methods

SLC26A4 was sequenced in 1056 Chinese patients with NSEVA. KCNJ10 was screened in 131 patients who lacked mutations in either one or both alleles of SLC26A4. Additionally, KCNJ10 was screened in 840 controls, including 563 patients diagnosed with NSEVA who carried biallelic SLC26A4 mutations, 48 patients with nonsyndromic hearing loss due to inner ear malformations that did not involve enlargement of the vestibular aqueduct (EVA), 96 patients with conductive hearing loss due to various causes, and 133 normal-hearing individuals with no family history of hereditary hearing loss.

Results

925 NSEVA patients were found carrying two-allele pathogenic SLC26A4 mutations. The most frequently detected KCNJ10 mutation was c.812G>A (p.R271H). Compared with the normal-hearing control subjects, the occurrence rate of c.812G>A in NSEVA patients with lacking mutations in one or both alleles of SLC26A4 had no significant difference(1.53% vs. 5.30%, χ² = 2.798, p = 0.172), which suggested that it is probably a nonpathogenic benign variant. KCNJ10 c.1042C>T (p.R348C), the reported EVA-related mutation, was not found in patients with NSEVA who lacked mutations in either one or both alleles of SLC26A4. Furthermore, the normal-hearing parents of patients with NSEVA having two SLC26A4 mutations carried the KCNJ10 c.1042C>T or c.812G>A mutation and a SLC26A4 pathogenic mutation.

Conclusion

SLC26A4 is the major genetic cause in Chinese NSEVA patients, accounting for 87.59%. KCNJ10 may not be a contributor to NSEVA in Chinese population. Other genetic or environmental factors are possibly play a role in the etiology of Chinese EVA patients with zero or monoallelic SLC26A4 mutation.     

The G to A transformation of rs4702 polymorphism in 3’UTR of FURIN reduced the risk of radiotherapy‐induced cognitive impairment in glioma patients

The G allele of rs4702 polymorphism has been reported to reduce the production of mature BDNF and FURIN, both of which were closely associated with cognitive functions. Real‐time PCR, ELISA and luciferase assay were performed to explore the interactions between miR‐338‐3p, FURIN and BDNF. T‐RFLP was used to assess the intestinal flora in the stool samples of glioma patients after radiotherapy. We grouped the 106 glioma patients recruited according to the rs4702 polymorphism. The results showed no obvious correlation between rs4702 polymorphism and the expression of miR‐338‐3p. However, rs4702‐A was associated with increased expression of FURIN and BDNF in the serum and PBMC of glioma patients after radiotherapy. Besides, the study found that rs4702‐A was remarkably associated with increased enterotype I and decreased enterotype III in the stool of glioma patients after radiotherapy. Rs4702‐A was also proved to be closely associated with increased MMSE, role functioning and social functioning at three months after radiotherapy. Furthermore, miR‐338‐3p repressed the expression of FURIN‐G. Compared with G allele, the presence of A allele of rs4702 polymorphism in FURIN could obstruct the suppressive effect of miR‐338‐3p upon the expression of FURIN and BDNF in intestinal flora. Therefore, the carriers of A allele will be challenged with less risk of radiotherapy‐induced cognitive impairment.     

An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult‐type granulosa cell tumors (AGCTs), introduces a target site for miR‐1236, which causes haploinsufficiency of the tumor‐suppressor FOXL2. This miR‐1236‐mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA‐loaded RNA‐induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR‐1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation‐mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA‐targeting disease‐associated mutations in the CDS by forming a non‐canonical miRISC.