Electroacupuncture at Sensitized Acupoints Relieves Somatic Referred Pain in Colitis Rats by Inhibiting Sympathetic-Sensory Coupling to Interfere with 5-HT Signaling Pathway.

OBJECTIVE: To investigate whether electroacupuncture (EA) at sensitized acupoints could reduce sympathetic-sensory coupling (SSC) and neurogenic inflammatory response by interfering with 5-hydroxytryptamine (5-HT)ergic neural pathways to relieve colitis and somatic referred pain, and explore the underlying mechanisms. METHODS: Rats were treated with 5% dextran sodium sulfate (DSS) solution for 7 days to establish a colitis model. Twelve rats were randomly divided into the control and model groups according to a random number table (n=6). According to the "Research on Rat Acupoint Atlas", sensitized acupoints and non-sensitized acupoints were determined. Rats were randomly divided into the control, model, Zusanli-EA (ST 36), Dachangshu-EA (BL 25), and Xinshu (BL 15) groups (n=6), as well as the control, model, EA, and EA + GR113808 (a 5-HT inhibitor) groups (n=6). The rats in the control group received no treatment. Acupuncture was administered on 2 days after modeling using the stimulation pavameters: 1 mA, 2 Hz, for 30 min, with sparse and dense waves, for 14 consecutive days. GR113808 was injected into the tail vein at 5 mg/kg before EA for 10 min for 7 consecutive days. Mechanical sensitivity was assessed with von Frey filaments. Body weight and disease activity index (DAI) scores of rats were determined. Hematoxylin and eosin staining was performed to observe colon histopathology. SSC was analyzed by immunofluorescence staining. Immunohistochemical staining was performed to detect 5-HT and substance P (SP) expressions. The calcitonin gene-related peptide (CGRP) in skin tissue and tyrosine hydroxylase (TH) protein levels in DRG were detected by Western blot. The levels of hyaluronic acid (HA), bradykinin (BK), prostaglandin I2 (PGI2) in skin tissue, 5-HT, tryptophan hydroxylase 1 (TPH1), serotonin transporters (SERT), 5-HT 3 receptor (5-HT3R), and 5-HT 4 receptor (5-HT4R) in colon tissue were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: BL 25 and ST 36 acupoints were determined as sensitized acupoints, and BL 15 acupoint was used as a non-sensitized acupoint. EA at sensitized acupoints improved the DAI score, increased mechanical withdrawal thresholds, and alleviated colonic pathological damage of rats. EA at sensitized acupoints reduced SSC structures and decreased TH and CGRP expression levels (P<0.05). Furthermore, EA at sensitized acupoints reduced BK, PGI2, 5-HT, 5-HT3R and TPH1 levels, and increased HA, 5-HT4R and SERT levels in colitis rats (P<0.05). GR113808 treatment diminished the protective effect of EA at sensitized acupoints in colitis rats (P<0.05). CONCLUSION: EA at sensitized acupoints alleviated DSS-induced somatic referred pain in colitis rats by interfering with 5-HTergic neural pathway, and reducing SSC inflammatory response.

Red cell distribution width-a potential prognostic indicator for colorectal cancer patients after radical resection in China.

Background: Red cell distribution width (RDW) can signal poor prognosis in inflammatory medical conditions. The purpose of the study was to investigate the relationship between preoperative RDW and colorectal cancer (CRC) in a large cohort of patients. Methods: A total of 6,224 CRC patients who underwent radical resection at the Fudan University Shanghai Cancer Center were evaluated retrospectively. The prognostic significance of RDW for overall survival (OS) and disease-free survival (DFS) was analyzed using Cox proportional hazards models and Kaplan-Meier method. Propensity score matching (PSM) was used based on survival confounding factors. Results: The mean age of the study participants was 59.5±12.0 years and the study cohort was 44% female. The overall median and mean RDW values were 13.3% and 14.0%, respectively. Patients were stratified into three groups based on their RDW value (≤13.3%, 13.4-14.0%, and >14.0%). OS and DFS were shown to significantly deteriorate with increasing RDW category. In the PSM population, OS and DFS were significantly lower in the high RDW group compared with matched controls. However, the differences vanished in the comparisons between the middle RDW group and the control group. Conclusions: Our findings demonstrate that preoperative RDW may represent a simple and powerful prognostic factor for CRC patients after radical resection. Integrating RDW into clinical practice may better inform the prognosis and optimize therapeutic approaches for patients with CRC.

Electroacupuncture Zusanli (ST36) Relieves Somatic Pain in Colitis Rats by Inhibiting Dorsal Root Ganglion Sympathetic-Sensory Coupling and Neurogenic Inflammation.

Referred somatic pain triggered by hyperalgesia is common in patients with inflammatory bowel disease (IBD). It was reported that sprouting of sympathetic nerve fibers into the dorsal root ganglion (DGR) and neurogenic inflammation were related to neuropathic pain, the excitability of neurons, and afferents. The purpose of the study was to explore the potential and mechanism of electroacupuncture (EA) at Zusanli (ST36) for the intervention of colon inflammation and hyperalgesia. Sprague-Dawley (SD) was randomly divided into four groups, including control, model, EA, and sham-EA. Our results showed EA treatment significantly attenuated dextran sulfate sodium- (DSS-) induced colorectal lesions and inflammatory cytokine secretion, such as TNF-α, IL-1ß, PGE2, and IL-6. EA also inhibited mechanical and thermal pain hypersensitivities of colitis rats. Importantly, EA effectively abrogated the promotion effect of DSS on ipsilateral lumbar 6 (L6) DRG sympathetic-sensory coupling, manifested as the sprouting of tyrosine hydroxylase- (TH-) positive sympathetic fibers into sensory neurons and colocalization of and calcitonin gene-related peptide (CGRP). Furthermore, EA at Zusanli (ST36) activated neurogenic inflammation, characterized by decreased expression of substance P (SP), hyaluronic acid (HA), bradykinin (BK), and prostacyclin (PGI2) in colitis rat skin tissues corresponding to the L6 DRG. Mechanically, EA treatment reduced the activation of the TRPV1/CGRP, ERK, and TLR4 signaling pathways in L6 DRG of colitis rats. Taken together, we presumed that EA treatment improved colon inflammation and hyperalgesia, potentially by suppressing the sprouting of sympathetic nerve fibers into the L6 DGR and neurogenic inflammation via deactivating the TRPV1/CGRP, ERK, and TLR4 signaling pathways.

Molecular docking and molecular dynamics study Lianhua Qingwen granules (LHQW) treats COVID-19 by inhibiting inflammatory response and regulating cell survival.

Purpose: 2019 Coronavirus disease (COVID-19) is endangering health of populations worldwide. Latest research has proved that Lianhua Qingwen granules (LHQW) can reduce tissue damage caused by inflammatory reactions and relieve patients' clinical symptoms. However, the mechanism of LHQW treats COVID-19 is currently lacking. Therefore, we employed computer simulations to investigate the mechanism of LHQW treats COVID-19 by modulating inflammatory response. Methods: We employed bioinformatics to screen active ingredients in LHQW and intersection gene targets. PPI, GO and KEGG was used to analyze relationship of intersection gene targets. Molecular dynamics simulations validated the binding stability of active ingredients and target proteins. Binding free energy, radius of gyration and the solvent accessible surface area were analyzed by supercomputer platform. Results: COVID-19 had 4628 gene targets, LHQW had 1409 gene targets, intersection gene targets were 415. Bioinformatics analysis showed that intersection targets were closely related to inflammation and immunomodulatory. Molecular docking suggested that active ingredients (including: licopyranocoumarin, Glycyrol and 3-3-Oxopropanoic acid) in LHQW played a role in treating COVID-19 by acting on CSF2, CXCL8, CCR5, NLRP3, IFNG and TNF. Molecular dynamics was used to prove the binding stability of active ingredients and protein targets. Conclusion: The mechanism of active ingredients in LHQW treats COVID-19 was investigated by computer simulations. We found that active ingredients in LHQW not only reduce cell damage and tissue destruction by inhibiting the inflammatory response through CSF2, CXCL8, CCR5 and IFNG, but also regulate cell survival and growth through NLRP3 and TNF thereby reducing apoptosis.

Early warning prevention and control strategies to reduce perioperative venous thromboembolism in patients with gastrointestinal cancer.

BACKGROUND: Venous thromboembolism (VTE) is a major cause of unexpected and perioperative in-hospital deaths. It is characterized by high morbidity, high mortality, high misdiagnosis rate, and high missed diagnosis rates. VTE is a common postoperative complication in cancer patients. VTE is preventable, and early identification of risk factors leading to VTE and appropriate early preventive actions can reduce its occurrence and mortality. Presently, there is no uniform standard for the prevention and control of VTE in clinical practice, and hospitals in China lack mature and effective protocols for the assessment, prevention, and treatment of VTE. AIM: To explore whether an early warning program could influence the occurrence of deep vein thrombosis (DVT) postoperatively. METHODS: This is a comparative retrospective cohort study, which enrolled patients who underwent laparotomic or laparoscopic gastrointestinal tumor resection for gastrointestinal cancer between January 2016 and December 2019. Patients were divided into a control group and an early warning group depending on whether or not the early warning program was implemented. A venous thromboembolism prevention and control team was established. The outcomes included the occurrence of DVT, the correct rate of VTE assessment, the coagulation indicators, and the mastery of VTE knowledge by the nurses. RESULTS: A total of 264 patients were included in this study, with 128 patients in the control group and 136 patients in the early warning group. The occurrence rate of DVT in the early warning group was 6.6% (9/136), compared with 14.1% (18/128) in the control group (P < 0.05). The correct rates of VTE risk assessment by the nurses and standard implementation rate of VTE preventive measures were 86.8% vs 65.6% and 80.2% vs 57.8% in early warning and control groups, respectively (all P < 0.001). The independent factors associated with postoperative DVT occurrence were age (OR = 1.083, 95%CI: 1.070-3.265, P = 0.032), Hyperlipidemia (OR = 1.127, 95%CI: 1.139-2.564, P = 0.042), preoperative high VTE risk (OR = 2.131, 95%CI: 1.085-5.178, P = 0.001), time of operation (OR = 2.268, 95%CI: 2.005-5.546, P = 0.026) and not adoption of early warning prevention (OR = 3.747, 95%CI: 1.523-6.956, P = 0.017). CONCLUSION: The early warning strategy was independently associated with the decreasing occurrence of VTE, and it might be suitable for protection from VTE in patients undergoing gastrointestinal cancer surgery.

Impact of operation duration on short-term and long-term prognosis in patients undergoing radical colorectal surgery.

Purpose: The objective of this study was to evaluate the impact of operation duration on short- and long-term outcomes of colorectal cancer patients following surgical resection. Methods: 6224 consecutive patients who underwent radical colorectal surgery were retrospectively assessed and were divided into short operation duration group (SOD) and long operation duration group (LOD) according to the operation duration cutoff value of 110 minutes. Results: Compared with patients in LOD group, patients in SOD group had significantly lower total costs in hospital, reduced expenses for drugs and antibiotics, shorter length of stay (LOS) in hospital and in the ICU. Moreover, 5-year overall survival (OS) and disease-free survival (DFS) for patients in the SOD group were markedly higher than for patients in the LOD group. Mutivariate regression analysis indicated that longer operation duration was associated with poor prognosis, with a hazard ratio of 1.004 (1.003, 1.005) for OS and 1.005 (1.003, 1.006) for DFS. Finally, surgeons' qualifications had meaningful correlation with operation duration (r= 0.450). Conclusions: Operation duration is an independent risk factor for patients' short-term and long-term outcome after radical colorectal surgery. Improve the surgical skills of the surgeon may shorten the operation duration, and further improve the outcome for patients.

KLK8 promotes the proliferation and metastasis of colorectal cancer via the activation of EMT associated with PAR1.

Kallikrein-related peptidase 8 (KLK8) acts as an oncogene or anti-oncogene in various tumours, and the abnormal expression of KLK8 is involved in the carcinogenesis of several tumours. However, the role of KLK8 in colorectal cancer (CRC) and the underlying mechanism remain largely unclear. In this study, the carcinogenic effect of KLK8 was determined via CCK-8 and colony formation assays in vitro and a xenograft model in nude mice in vivo. The metastasis-promoting effect of KLK8 was investigated with transwell migration and invasion assays and wound-healing assay in vitro and a metastasis model in nude mice in vivo. Bioinformatics analyses and mechanistic experiments were conducted to elucidate the molecular mechanism. Herein, we reported that KLK8 had a promotive effect on the proliferation, migration and invasion of RKO and SW480 cells. Epithelial-mesenchymal transition (EMT) played an important role in the promotive effects of KLK8 on CRC. In addition, protease-activated receptor-1 (PAR-1) antagonist SCH79797 but not protease-activated receptor-2 (PAR-2) antagonist FSLLRY-NH2 attenuated the proliferation, migration and invasion of KLK8-upregulated RKO and SW480 cells. PAR-1 antagonist SCH79797 reduced the tumour volume of xenograft model and decreased the metastatic nodules in the livers of metastasis model. Furthermore, SCH79797 could reverse the positive impact of KLK8 on the EMT process in CRC both in vitro and in vivo. Taken together, these findings demonstrated for the first time that KLK8 promoted EMT and CRC progression, and this effect might be, at least partly mediated by PAR1-dependent pathway.

Upregulation of KLK8 Predicts Poor Prognosis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a growing cause of cancer-related mortality worldwide. Kallikrein-related peptidase 8 (KLK8) has potential clinical values in many cancers. However, the clinicopathological significances of KLK8 in PDAC remain unknown. We explored the relationship of KLK8 to clinicopathological features of PDAC based on public databases. KLK8 expression was examined in human PDAC tissues. Cell proliferation and apoptosis were evaluated in KLK8-overexpressed human pancreatic cancer cell lines Mia-paca-2 and Panc-1. The related signaling pathways of KLK8 involved in pancreatic cancer progression were analyzed by gene set enrichment analysis (GSEA) and further verified in in vitro studies. We found that KLK8 was up-regulated in tumor tissues in the TCGA-PAAD cohort, and was an independent prognostic factor for both overall survival and disease-free survival of PDAC. KLK8 mRNA and protein expressions were increased in PDAC tissues compared with para-cancerous pancreas. KLK8 overexpression exerted pro-proliferation and anti-apoptotic functions in Mia-paca-2 and Panc-1 cells. GSEA analysis showed that KLK8 was positively associated with PI3K-Akt-mTOR and Notch pathways. KLK8-induced pro-proliferation and anti-apoptotic effects in Mia-paca-2 and Panc-1 cells were attenuated by inhibitors for PI3K, Akt, and mTOR, but not by inhibitor for Notch. Furthermore, overexpression of KLK8 in Mia-paca-2 and Panc-1 cells significantly increased epidermal growth factor (EGF) levels in the culture media. EGF receptor (EGFR) inhibitor could block KLK8-induced activation of PI3K/Akt/mTOR pathway and attenuate pro-proliferation and anti-apoptotic of KLK8 in Mia-paca-2 and Panc-1 cells. In conclusion, KLK8 overexpression exerts pro-proliferation and anti-apoptotic functions in pancreatic cancer cells via EGF signaling-dependent activation of PI3K/Akt/mTOR pathway. Upregulated KLK8 in PDAC predicts poor prognosis and may be a potential therapeutic target for PDAC.

Dynamic changes of plasma extracellular vesicle long RNAs during perioperative period of colorectal cancer.

Extracellular vesicles (EVs) long RNAs (exLRs) have been shown to be indicators for the diagnosis and prognosis of colorectal cancer (CRC); however, the dynamic changes of exLRs during perioperative period and their cellular sources in CRC remains largely unknown. In this study, exLR sequencing (exLR-seq) was performed on plasma samples from three CRC patients at four time points (before surgery [T0], after extubation [T1], 1 day after surgery [T2], and 3 days after surgery [T3]). Bioinformatics approaches were used to investigate the profile and biofunctions of exLRs and their cellular sources. Greater than 12,000 mRNAs and 2,000 lncRNAs were reliably detected in each exLR-seq sample. Compared with T0, there were 110 differentially expressed genes (DEGs) in T1, 60 DEGs in T2, and 50 DEGs in T3. A total of 11 DEGs were found at all three time points and were related to membrane potential. In addition, compared to T0, 22 differentially expressed lncRNAs (DELRs) were found in T1, 19 DELRs in T2, and 38 DELRs in T3. Moreover, only three DELRs were detected at all three time points. Interestingly, EVs from CD8 + T cells, CD4+ memory T cells and NK cells decreased after surgery and the absolute quantity of EVs from immune cells were reduced as well. In summary, this study was the first to characterize the dynamic changes of exLRs during perioperative period and the cellular sources. These findings established the foundation for further studies involving the effects of these dynamically changed exLRs on CRC.

SET8 suppression mediates high glucose-induced vascular endothelial inflammation via the upregulation of PTEN.

Hyperglycemia-mediated endothelial inflammation participates in the pathogenesis of cardiovascular complications in subjects with diabetes. Previous studies reported that phosphatase and tensin homolog deleted on chromosome ten (PTEN) and SET8 participate in high glucose-mediated endothelial inflammation. In this study, we hypothesize that SET8 regulates PTEN expression, thus contributing to high glucose-mediated vascular endothelial inflammation. Our data indicated that plasma soluble intercellular adhesion molecule-1 (sICAM-1) and endothelial selectin (e-selectin) were increased in patients with diabetes and diabetic rats. PTEN expression was augmented in the peripheral blood mononuclear cells of patients with diabetes and in the aortic tissues of diabetic rats. Our in vitro study indicated that high glucose increased monocyte/endothelial adhesion, endothelial adhesion molecule expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose led to endothelial inflammation via upregulation of PTEN. Furthermore, high glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. SET8 overexpression reversed the effects of high-glucose treatment. shSET8-mediated endothelial inflammation was counteracted by siPTEN. Furthermore, SET8 was found to interact with FOXO1. siFOXO1 attenuated high glucose-mediated endothelial inflammation. FOXO1 overexpression-mediated endothelial inflammation was counteracted by siPTEN. H4K20me1 and FOXO1 were enriched in the PTEN promoter region. shSET8 increased PTEN promoter activity and augmented the positive effect of FOXO1 overexpression on PTEN promoter activity. Our in vivo study indicated that SET8 was downregulated and FOXO1 was upregulated in the peripheral blood mononuclear cells of patients with diabetes and the aortic tissues of diabetic rats. In conclusion, SET8 interacted with FOXO1 to modulate PTEN expression in vascular endothelial cells, thus contributing to hyperglycemia-mediated endothelial inflammation.

High glucose mediates NLRP3 inflammasome activation via upregulation of ELF3 expression.

Microtubule affinity regulating kinase 4 (MARK4) plays a crucial role in the regulation of NOD-like receptor pyrin domain 3 (NLRP3) inflammasome activation, which leads to the generation of bioactive interleukin (IL)-1ß and IL-18. E74-like ETS transcription factor 3 (ELF3) participates in endothelial inflammatory processes. We hypothesized that ELF3 modulates MARK4 expression in vascular endothelial cells, thus contributing to high glucose-mediated NLRP3 inflammasome activation. Plasma IL-1ß, IL-18, NLRP3 inflammasome and MARK4 expression was increased in diabetic patients and rats. An in vitro study indicated that high glucose increased IL-1ß and IL-18 expression and activated the NLRP3 inflammasome via upregulation of MARK4 in human umbilical vein endothelial cells (HUVECs). Furthermore, high glucose increased ELF3 expression. ELF3 downregulation reversed the effects of high glucose treatment. Accordingly, the effects of ELF3 overexpression were similar to those of high glucose treatment and were counteracted by siMARK4. Furthermore, ELF3 was found to interact with SET8. High glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. Overexpression of SET8 inhibited high glucose-induced MARK4 expression and NLRP3 inflammasome activation. The effects of shSET8 were similar to those of high glucose treatment and were counteracted by siMARK4. A mechanistic study found that ELF3 and H4K20me1 were enriched in the MARK4 promoter region. si-ELF3 attenuated MARK4 promoter activity and augmented the inhibitory effect of SET8 on MARK4 promoter activity. Furthermore, SET8 downregulation and ELF3 upregulation were confirmed in diabetic patients and rats. In conclusion, ELF3 interacted with SET8 to modulate MARK4 expression, which participated in hyperglycaemia-mediated endothelial NLRP3 inflammasome activation.

Propofol attenuates TNF-α-induced MMP-9 expression in human cerebral microvascular endothelial cells by inhibiting Ca2+/CAMK II/ERK/NF-κB signaling pathway.

Metalloproteinase 9 (MMP-9) is able to degrade collagen IV, an important component of blood-brain barrier (BBB). Expression of MMPs, especially MMP-9, correlates with BBB disruption during central nervous system inflammation. Propofol has been reported to have anti-inflammation effects. In this study, we investigated the effects of propofol on TNF-α-induced MMP-9 expression in human cerebral microvascular endothelial cells (hCMEC/D3 cells) and explored the underlying mechanisms. The hCMEC/D3 cells were treated with propofol (25 µM), followed by TNF-α (25 ng/mL). We showed that TNF-α treatment markedly increased MMP-9 expression and decreased collagen IV expression in hCMEC/D3 cells, which was blocked by pretreatment with propofol. TNF-α-induced downregulation of collagen IV was also reversed by MMP-9 knockdown with siRNA. We revealed that TNF-α upregulated MMP-9 expression in hCMEC/D3 cells through activation of Ca2+/CAMK II/ERK/NF-κB signaling pathway; co-treatment with inhibitors of CaMK II (KN93), ERK (LY3214996), NF-κB (PDTC) or Ca2+chelator (BAPTA-AM) abrogated the effect of TNF-α on MMP-9 expression. We further established an in vitro BBB model by co-culturing of hCMEC/D3 cells and human astrocytes for 6 days and measuring trans-endothelial electrical resistance (TEER) to reflect the BBB permeability. TNF-α treatment markedly decreased TEER value, which was attenuated by pretreatment with propofol (25 µM) or MMP-9 knockdown with siRNA. In conclusion, propofol inhibits TNF-α-induced MMP-9 expression in hCMEC/D3 cells via repressing the Ca2+/CAMKII/ERK/NF-κB signaling pathway. TNF-α-impaired BBB integrity could be reversed by propofol, and propofol attenuates the inhibitory effect of TNF-α on collagen IV.

Characterization of factors underlying the metabolic shifts in developing kernels of colored maize.

Elucidation of the metabolic pathways determining pigmentation and their underlying regulatory mechanisms in maize kernels is of high importance in attempts to improve the nutritional composition of our food. In this study, we compared dynamics in the transcriptome and metabolome between colored SW93 and white SW48 by integrating RNA-Seq and non-targeted metabolomics. Our data revealed that expression of enzyme coding genes and levels of primary metabolites decreased gradually from 11 to 21 DAP, corresponding well with the physiological change of developing maize kernels from differentiation through reserve accumulation to maturation, which was cultivar independent. A remarkable up-regulation of anthocyanin and phlobaphene pathway distinguished SW93 from SW48, in which anthocyanin regulating transcriptional factors (R1 and C1), enzyme encoding genes involved in both pathways and corresponding metabolic intermediates were up-regulated concurrently in SW93 but not in SW48. The shift from the shikimate pathway of primary metabolism to the flavonoid pathway of secondary metabolism, however, appears to be under posttranscriptional regulation. This study revealed the link between primary metabolism and kernel coloration, which facilitate further study to explore fundamental questions regarding the evolution of seed metabolic capabilities as well as their potential applications in maize improvement regarding both staple and functional foods.

Phytoremediation and phytosensing of chemical contaminant, toluene: identification of the required target genes.

As an industrial chemical produced worldwide in high volumes, toluene is commonly detected in ambient air and water. It can combine with oxygen and form compounds that are harmful to humans. In recent years, phytoremediation has been increasingly applied to repair the environmental damage caused by pollutants. However, insufficient knowledge is available regarding the response of plants to toluene. To detect the potential genes in plants that are related to the sensing mechanism and metabolism of toluene, a microarray analysis has been conducted on Arabidopsis thaliana seedlings grown on toluene-containing media. Following the validation of data and the application of appropriate selection criteria, the results show a coordinated induction and suppression of 202 and 67 toluene-responsive genes, respectively. Within the functional class "metabolism", the genes encoding detoxification proteins represent the most strongly up-regulated group. These include genes encoding cytochrome P450s, glucosyl transferases, and transporters. Subsequently, the toluene-induced genes of Arabidopsis are analyzed in detail.