Calcaneal fractures: 3D-printing model to assist spatial weaving of percutaneous screws versus conventional open fixation-a retrospective cohort study.

PURPOSE: This study compared the clinical effectiveness and wound complications of a three-dimensional model-assisted spatial weaving screw fixation (3D-SWSF) versus open reduction and internal fixation (ORIF) via an L-shaped extensile lateral approach for calcaneal fractures. METHODS: This single-centre retrospective cohort study was conducted with two cohort groups in which patients with Sanders II and III calcaneal fractures underwent 3D-SWSF or conventional ORIF. The clinical outcome measures included operation duration, time to operation, wound complications, blood loss volume, hospital stays, American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot score, and visual analog scale (VAS) score. The pre-operative, post-operative, and one-year follow-up Gissane's angle (GA), Böhler's angle (BA), height, and width and length of the calcaneal fractures were also compared between the two groups. RESULTS: From Oct 2015 to Oct 2019, 31 patients received 3D-SWSF and 41 received conventional ORIF. A total of 11 (26.8%) patients in ORIF group had wound complications, compared with only two (6.5%) in 3D-SWSF group (p = 0.032). Operative time, blood loss, and hospital stay in 3D-SWSF group were lesser than those in ORIF group. The patients treated with 3D-SWSF had better AOFAS and VAS scores than those treated with ORIF at the last follow-up. The post-operative and one-year follow-up radiographic indexes as well as the GA, BA, length, width, and height of the calcaneal fractures were relatively comparable between the two groups. CONCLUSION: Our study revealed that 3D-SWSF could effectively decrease the risk of wound complications, shorten operation time, reduce length of hospitalization, and improve post-operative rehabilitation.

Design and Biomechanical Finite Element Analysis of Spatial Weaving Infracalcaneal Fixator System.

OBJECTIVE: Traditional internal fixation of calcaneus fractures, involving lateral L-shaped incisions and plate fixation, has disadvantages such as increased operative exposure, eccentric plate fixation, and complications. The aim of this study was to design a Spatial Weaving Intra-calcaneal Fixator System (SWIFS) for the treatment of complex calcaneal fractures and to compare its biomechanical properties with those of traditional calcaneal plates. METHODS: The computed tomography (CT) data of the normal adult calcaneus was used for modeling, and the largest trapezoidal column structure was cut and separated from the model and related parameters were measured. The SWIFS was designed within the target trapezoid, according to the characteristics of the fracture of the calcaneus. The Sanders model classification type IV calcaneal fracture was established in finite element software, and fixation with calcaneal plate and the SWIFS examined. Overall structural strength distribution and displacement in the two groups were compared. RESULTS: The maximum 3D trapezoidal column in the calcaneus was constructed, and the dimensions were measured. The SWIFS and the corresponding guide device were successfully designed. In the one-legged erect position state, the SWIFS group exhibited a peak von Mises equivalent stress of 96.00 MPa, a maximum displacement of 0.31 mm, and a structural stiffness of 2258.06 N/mm. The conventional calcaneal plate showed a peak von Mises equivalent stress of 228.66 Mpa, a maximum displacement of 1.26 mm, and a structural stiffness of 555.56 N/mm. The SWIFS group exhibited a 75.40% decrease in displacement and a 306.45% increase in stiffness. CONCLUSION: Compared with fixation by conventional calcaneal plate, the SWIFS provides better structural stability and effective stress distribution.

Bioinformatics to analyze the differentially expressed genes in different degrees of Alzheimer's disease and their roles in progress of the disease.

Employing bioinformatics approaches, this investigation pinpointed pivotal differentially expressed genes (DEGs) across the spectrum of Alzheimer's disease (AD), from incipient to severe stages, using the GSE28146 dataset from the GEO repository. Analytical methods included DEG identification via the limma package in R, coupled with GO and KEGG pathway analyses through clusterProfiler, to discern biological processes and pathway involvements. Key findings spotlighted the roles of proteasome subunits PSMB4, PSMB8, PSMC4, and PSMD6 in the early stage, ribosomal proteins RPS3 and RPL11 during moderate AD, and mitochondrial components COX5B, COX6B2, and COX7A2 in severe AD, underscoring their importance in the disease's pathogenesis. Conclusively, these results not only delineate the dynamic genetic shifts accompanying AD progression but also propose critical biomarkers for potential therapeutic targeting, offering a consolidated basis for future AD research and treatment development. This offered a novel idea for analyzing the pathogenesis and development of AD and investigation of targeted drugs.

Memantine alleviates cognitive impairment and hippocampal morphology injury in a mouse model of chronic alcohol exposure.

Alcohol-related cognitive impairment (ARCI) is highly prevalent among patients with alcohol abuse and dependence. The pathophysiology of ARCI, pivotal for refined therapeutic approaches, is not fully elucidated, posing a risk of progression to severe neurological sequelae such as Korsakoff's syndrome (KS) and Alcohol-Related Dementia (ARD). This study ventures into the underlying mechanisms of chronic alcohol-induced neurotoxicity, notably glutamate excitotoxicity and cytoskeletal disruption, and explores the therapeutic potential of Memantine, a non-competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor known for its neuroprotective effect against excitotoxicity. Our investigation centers on the efficacy of Memantine in mitigating chronic alcohol-induced cognitive and hippocampal damages in vivo. Male C57BL/6J mice were subjected to 30 % (v/v, 6.0 g/kg) ethanol via intragastric administration alongside Memantine co-treatment (10 mg/kg/day, intraperitoneally) for six weeks. The assessment involved Y maze, Morris water maze, and novel object recognition tests to evaluate spatial and recognition memory deficits. Histopathological evaluations of the hippocampus were conducted to examine the extent of alcohol-induced morphological changes and the potential protective effect of Memantine. The findings reveal that Memantine significantly improves chronic alcohol-compromised cognitive functions and mitigates hippocampal pathological changes, implicating a moderating effect on the disassembly of actin cytoskeleton and microtubules in the hippocampus, induced by chronic alcohol exposure. Our results underscore Memantine's capability to attenuate chronic alcohol-induced cognitive and hippocampal morphological harm may partly through regulating cytoskeleton dynamics, offering valuable insights into innovative therapeutic strategies for ARCI.

Prediction of Rhizoma Drynariae Targets in the Treatment of Osteonecrosis of the Femoral Head based on Network Pharmacology and Experimental Verification.

BACKGROUND: Rhizoma drynariae, a classic prescription in traditional Chinese medicine, has long been used for the treatment of osteonecrosis of the femoral head (ONFH), but its potential targets and molecular mechanisms remain to be further explored. OBJECTIVE: This study aims to explore the mechanism of Rhizoma drynariae in ONFH treatment via network pharmacology and in vitro experiments. METHODS: Targets of Rhizoma drynariae and ONFH were predicted using relevant databases, and intersection analysis was conducted to screen for shared targets. A PPI network of the shared targets was built using STRING to identify the key targets. Functional enrichment analyses of Gene Ontology and KEGG pathway data were carried out using R software. The compound-target-pathway network was constructed for Rhizoma Drynariae in the treatment with ONFH using Cytoscape 3.9.0. Cell proliferation was assessed using CCK8 and apoptosis was detected using (Propidium Iodide) PI staining and western blotting. RESULTS: This study depicts the interrelationship of the bioactive compounds of Rhizoma drynariae with ONFH-associated signaling pathways and target receptors and is a potential reagent for ONFH treatment. CONCLUSION: Based on a network pharmacology analysis and in vitro experiment, we predicted and validated the active compounds and potential targets of Rhizoma drynariae, provide valuable evidence of Rhizoma Drynariae in future ONFH treatment.

Clinical Diagnostic Values of Transfer RNA-Derived Fragment tRF-41-YDLBRY73W0K5KKOVD and its Effects on the Growth of Gastric Cancer Cells.

Gastric cancer (GC) is a serious disease with high mortality and poor prognosis. It is known that tRNA halves play key roles in the progression of cancer. This study explored the function of the tRNA half tRF-41-YDLBRY73W0K5KKOVD in GC. Quantitative real-time reverse transcription-polymerase chain reaction was used to measure RNA levels. The level of tRF-41-YDLBRY73W0K5KKOVD in GC cells was regulated by its mimics or inhibitor. Cell proliferation was evaluated by using a Cell Counting Kit-8 and EdU cell proliferation assay. A Transwell assay was used to detect cell migration. Flow cytometry was used to measure cell cycle and apoptosis. The results showed that tRF-41-YDLBRY73W0K5KKOVD expression was decreased in GC cells and tissues. Functionally, overexpression of tRF-41-YDLBRY73W0K5KKOVD inhibited cell proliferation, reduced migration, repressed the cell cycle, and promoted cell apoptosis in GC cells. Based on RNA sequencing results and luciferase reporter assays, 3'-phosphoadenosine-5'-phosphosulfate synthase 2 (PAPSS2) was identified as a target gene of tRF-41-YDLBRY73W0K5KKOVD. These findings indicated that tRF-41-YDLBRY73W0K5KKOVD inhibited GC progression, suggesting that tRF-41-YDLBRY73W0K5KKOVD might be a potential therapeutic target in GC.

Screening of Crucial Differentially-Methylated/Expressed Genes for Alzheimer's Disease.

Background: We aimed to make an integrated analysis of published transcriptome and DNA methylation dataset to ascertain the key differentially methylated and differentially expressed genes for Alzherimer's disease (AD). Methods: Two gene expression microarrays and 1 gene methylation microarray were downloaded for identification of differentially expressed genes and differentially methylated genes. Then, we used various biological information databases to annotate the functions of the differentially-methylated/expressed genes, and screen out key genes and important signaling pathways. Finally, we validate the differentially-methylated/expressed genes in the additional online datasets and in blood from AD patients.Results: A total of 8 hub hypomethylated-high expression genes were obtained, including Rac family small GTPase 2, FGR proto-oncogene, Src family tyrosine kinase, LYN proto-oncogene, Src family tyrosine kinase, protein kinase C delta, myosin IF, integrin subunit alpha 5, semaphorin 4D, and growth arrest specific protein 7. Some enriched signaling pathways of hypomethylated-high expression genes were identified, including regulation of actin cytoskeleton, chemokine signaling pathway, Fc gamma R-mediated phagocytosis, and axon guidance. Conclusion: Differentially-methylated/expressed genes are likely to be associated with AD.

Ginkgetin promotes proliferation and migration of Schwann cells via PIGF/p38 MAPK signaling pathway.

The proliferation and migration of Schwann cells is pivotal to peripheral nerve injury (PNI) repair. Recent studies have revealed that Ginkgetin has neuroprotective effects. Hence, we focused on identifying whether Ginkgetin could regulate the proliferation and migration of Schwann cells, thereby contributing to the repair of PNI. Rat Schwann cells RSC96 were treated with different concentrations of Ginkgetin. Short hairpin RNA targeting phosphatidylinositol glycan anchor biosynthesis class F (shPIGF) was employed to investigate the effects of PIGF on Ginkgetin-induced RSC96 cells. Viability of RSC96 cells was estimated via cell counting kit-8 (CCK-8) assay and proliferation of the cells was assessed by 5-ethynyl-2'-deoxyuridine (EdU) assay. Migration was estimated via wound healing assay and invasion was evaluated through transwell assay. Western blot was employed to test the expressions of PIGF, protein-38 (p38), and phosphorylated p-38 (p-p38). Ginkgetin (50 or 100 µg/ml) increased the viability, proliferation, migration, and invasion of RSC96 cells, up-regulated PIGF expression and raised the ratio of p-p38/p38, which were all reversed by PIGF silencing. Ginkgetin promotes proliferation, migration, and invasion of Schwann cells via PIGF/p38 MAPK signaling pathway.

Characteristics and Predictors of Long-Time Survivors in Non-Metastatic Gastric Signet Ring Cell Carcinoma: A Large Population-Based Study.

Objective: Gastric signet ring cell carcinoma (SRCC) is a distinct entity with a relatively poor prognosis. This study analyzed the clinicopathological characteristics of long-time survivors (LTSs) and identified independent predictors of long-term survival (LTS) in non-metastatic gastric SRCC. Methods: Data from 3906 patients with non-metastatic gastric SRCC were retrieved from Surveillance, Epidemiology and End Results according to the inclusion and exclusion criteria. Patients were randomly divided into training and validation cohorts. Predictors of LTS in the training cohort were identified by multivariate logistic regression. A nomogram-based predictive model for LTS was constructed in non-metastatic gastric SRCC. Results: There were 800 patients who survived for >5 years and were defined as TLSs. Young age, other race (not black or white population), female gender, married status, small tumor size, low tumor infiltration, and negative lymph node involvement were independent predictors of LTS in non-metastatic gastric SRCC. These seven variables were incorporated into a nomogram model for predicting LTS. The calibration curve showed good consistency between observed and predicted probability of LTS, and the receiver operating characteristic curve showed acceptable discriminative capacity in the training and validation cohorts. Conclusion: This study provides an overview of the features of patients with non-metastatic gastric SRCC. Age, race, sex, marital status, tumor size, tumor infiltration, and lymph node involvement were identified as independent predictors of LTS.

tRNA-derived small RNAs: Mechanisms and potential roles in cancers.

Transfer RNAs (tRNAs) are essential for protein synthesis. Mature or pre-tRNAs may be cleaved to produce tRNA-derived small RNAs (tsRNAs). tsRNAs, divided into tRNA-derived stress-induced RNA (tiRNAs) and tRNA-derived fragments (tRFs), play versatile roles in a number of fundamental biological processes. tsRNAs not only play regulatory roles in gene silencing, RNA stability, reverse transcription, and translation, but are also closely related to cell proliferation, migration, cell cycle, and apoptosis. Their abnormal expression is associated with the occurrence and development of various human diseases, especially cancer. This paper reviews the classification, biogenesis, and mechanism of action of tsRNAs, and the research progress to date on tsRNAs in cancers. These findings provide new opportunities for diagnostic biomarkers and treatment targets of several types of cancers including gastric cancer, colorectal cancer, hepatocellular carcinomas, pancreatic cancer, breast cancer, prostate cancer, renal cell carcinoma, ovarian cancer, lung cancer, bladder cancer, thyroid cancer, oral cancer, and leukemia.

Ezetimibe Prevents IL-1ß-induced Inflammatory Reaction in Mouse Chondrocytes via Modulating NF-κB and Nrf2/HO-1 Signaling Crosstalk.

BACKGROUND: Osteoarthritis is a type of age-related, chronic, and degenerative joint disease. Ezetimibe, a cholesterol absorption inhibitor, is widely used for the treatment of various diseases. However, the role of ezetimibe in osteoarthritis remains unclear. OBJECTIVES: This study aimed to explore the anti-inflammation effect of ezetimibe on mouse chondrocytes. METHODS: In the present study, ELISA, qPCR and western blot analysis were performed to evaluate the anti-inflammatory effects of ezetimibe. In addition, enzymes that are highly associated with the anabolism and catabolism of the extracellular matrix of the articular cartilage were also evaluated. RESULTS: Treatment with ezetimibe attenuated the IL-1ß-induced degradation of the extracellular matrix, including aggrecan and collagen II. Ezetimibe also attenuated the IL-1ß-induced expression levels of MMP3, MMP13 and ADAMTS5, thus exerting protective effects against IL-1ß- induced extracellular matrix degradation. The complex mechanism of the anti-inflammatory reaction contributed to the activation of the Nrf2/HO-1 pathway and the suppression of the NF-κB pathway. CONCLUSION: On the whole, the present study demonstrates that ezetimibe may be a promising agent for further osteoarthritis therapy.

Extracellular vesicles-associated tRNA-derived fragments (tRFs): biogenesis, biological functions, and their role as potential biomarkers in human diseases.

Traditionally, transfer RNAs (tRNAs) specifically decoded messenger RNA (mRNA) and participated in protein translation. tRNA-derived fragments (tRFs), also known as tRNA-derived small RNAs (tsRNAs), are generated by the specific cleavage of pre- and mature tRNAs and are a class of newly defined functional small non-coding RNAs (sncRNAs). Following the different cleavage positions of precursor or mature tRNA, tRFs are classified into seven types, 5'-tRNA half, 3'-tRNA half, tRF-1, 5'U-tRF, 3'-tRF, 5'-tRF, and i-tRF. It has been demonstrated that tRFs have a diverse range of biological functions in cellular processes, which include inhibiting protein translation, modulating stress response, regulating gene expression, and involvement in cell cycles and epigenetic inheritance. Emerging evidences have indicated that tRFs in extracellular vesicles (EVs) seem to act as regulatory molecules in various cellular processes and play essential roles in cell-to-cell communication. Furthermore, the dysregulation of EV-associated tRFs has been associated with the occurrence and progression of a variety of cancers and they can serve as novel potential biomarkers for cancer diagnosis. In this review, the biogenesis and classification of tRFs are summarized, and the biological functions of EV-associated tRFs and their roles as potential biomarkers in human diseases are discussed.

Effectiveness of Physical Activity Interventions on Cognition, Neuropsychiatric Symptoms, and Quality of Life of Alzheimer's Disease: An Update of a Systematic Review and Meta-Analysis.

The topic of physical activity interventions for the treatment of Alzheimer's disease (AD) has been discussed for decades, but there are still inconsistent views on the effect of its intervention in different studies. With the increase in randomized controlled trials (RCTs), it is necessary to update newly published studies and systematically evaluate the effects of physical activity interventions. Scientific citation databases (e.g., PubMed, EMBASE, etc.) and registration databases (e.g., ISRCTN, CHICTR, etc.) were checked to screen RCTs and systematic reviews of physical activity interventions in AD. Then extract and review the intervention methods and their evaluation results in the included studies. Spearman correlation method was used to test the association between the mean difference (MD) of intervention results and activity time. The Hedges'g method was used to combine continuous data to analyze the standard MD (SMD) of different intervention types or time subgroups. The overall results show that physical activity intervention can improve the cognition, neuropsychiatric symptoms and quality of life (Qol) of AD patients, but the duration of the intervention significantly affected the outcome of the assessment. Subgroup analysis results showed that an intervention duration of 2-5 months had a significant advantage: cognitive function (Minimum Mental State Examination: SMD = 0.47, 95% CI = 0.33 ∼ 0.61, P < 0.01), neuropsychiatric symptoms (Neuropsychiatric Inventory: SMD = -0.48, 95% CI = -0.85 ∼-0.11, P < 0.01), and quality of life (Qol-AD: SMD = 0.47, 95% CI = 0.23 ∼ 0.71, P < 0.01). The systematic review and analysis results of updated RCTs suggested that short-term (2-5 months) physical activity interventions were more beneficial in improving cognitive function, neuropsychiatric symptoms and Qol in patients with AD. And there was no evidence of differences in the effectiveness of different physical activity interventions.

Effects of biophilic virtual reality on cognitive function of patients undergoing laparoscopic surgery: study protocol for a sham randomised controlled trial.

INTRODUCTION: Virtual reality (VR) is already being used for cognitive or emotional rehabilitation. However, its role in postoperative cognitive dysfunction (POCD) has not been fully recognised. Due to the lack of an effective postoperative follow-up system, the incidence of POCD in China is not clear, and although many drugs have been proposed to improve POCD in the animal study, their clinical applications are limited, while VR provides an innovative method to provide non-pharmacological management. METHODS AND DESIGN: This is a single-centre, randomised, double-blind, sham-controlled clinical trial. In this study, 600 patients over 55 years old undergoing laparoscopic surgery will be recruited. Participants will be randomly assigned to receive biophilic VR or sham VR (1:1 ratio), all patients have 20 min of exposure per day during the hospital stay. The primary outcome is the impact of VR on the incidence of POCD. Secondary outcomes include perioperative anxiety and instrumental activities of daily living. Changes in the performance of the neurocognitive batteries are measured by a local resident doctor. Serum samples will be collected on the day before surgery and 7 days after surgery. ETHICS AND DISSEMINATION: This trial has ethical approval from the Medical Ethics Committee of the Affiliated Hospital of Medical School of Ningbo University (KY20210302). The study is sponsored by Ningbo University and Ningbo Science and Technology Bureau. CONTACT: Dr. Mao Haijiao, Chair of the hospital medical Ethics committee (ndfylunli@126.com). Trial results will be submitted for publication in peer-reviewed journals, patient recruitment began in April 2021. Written informed consent is obtained for all participants. All information acquired will be disseminated via national or international conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ChiCTR2000040919.

Oroxylin A attenuates IL-1ß-induced inflammatory reaction via inhibiting the activation of the ERK and PI3K/AKT signaling pathways in osteoarthritis chondrocytes.

Osteoarthritis (OA) is characterized by degradation of the articular cartilage, synovium inflammation, subchondral bone sclerosis and osteophyte formation. OA is the most common degenerative joint disorder among the elderly population. In particular, currently available therapeutic strategies, such as non-steroidal anti-inflammatory drugs (NSAIDs) may cause severe side-effects. Therefore, novel candidate targets for OA therapy are urgently needed. Oroxylin A (OrA) is a natural mono-flavonoid that can be extracted from Scutellariae radix. The present study aimed to investigate the potential effects of OrA on interleukin (IL)-1ß-induced chondrocytes inflammatory reactions. The current study performed quantitative PCR, western blotting and cell immunofluorescence to evaluate the effect of Oroxylin A in chondrocyte inflammation. The results demonstrated that OrA significantly attenuated the upregulation of inducible nitric oxide synthase and cyclooxygenase 2 by IL-1ß at both protein and mRNA levels. IL-1ß-stimulated upregulation of matrix metalloproteinase (MMP)-3 and MMP-13 expression, in addition to disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 expression, were all inhibited by OrA. Treatment with OrA significantly reversed the degradation of type II collagen and aggrecan by IL-1ß. Mechanistically, OrA suppressed the IL-1ß induced activation of ERK1/2 and PI3K/AKT signaling pathways. In conclusion, these findings suggest that OrA can serve as a potential therapeutic agent for the treatment of OA.

Morusin Inhibits Human Osteosarcoma via the PI3K-AKT Signaling Pathway.

OBJECTIVE: Osteosarcoma is considered as one of the most common types of bone tumors, which occurs among adolescents and children. METHODS: Current therapy strategies still have limited effectiveness therefore, the development of new therapies is urgent. Morusin is a compound isolated from Morus australis (Moraceae). Many studies have reported its anti-tumor effect on several tumor types. However, its role in osteosarcoma is still unclear. RESULTS: In this study, we determined that morusin significantly suppresses the proliferation and promotes the apoptosis of osteosarcoma cells. Furthermore, the migration and invasion of osteosarcoma were reduced after exposure to morusin. The deep mechanism was determined to be the inhibition of the PI3K/AKT signaling pathway. CONCLUSION: In conclusion, our study indicates morusin as a potential candidate for osteosarcoma therapy.

Alantolactone suppresses human osteosarcoma through the PI3K/AKT signaling pathway.

Osteosarcoma is the most common type of malignant bone cancer and results in cancer­related deaths among adolescents. Alantolactone (ALT) demonstrates antitumor properties in various diseases; however, its potential role in osteosarcoma is relatively unclear. The aim of the present study was to evaluate the effect of ALT on osteosarcoma. ALT significantly decreased the viability of U2OS and HOS osteosarcoma cell lines. Cells flow cytometry assay and Hoechst 33258 staining assay revealed that ALT significantly increased the proportion of apoptotic U2OS cells. In addition, wound healing and Transwell invasion assays demonstrated that the invasion and migration of osteosarcoma were markedly reduced upon ALT treatment. It was hypothesized that the antitumor functions of ALT are mediated through inhibition of the PI3K/AKT signaling pathway. In conclusion, the results of the present study confirmed the inhibition of ALT on osteosarcoma cells via downregulation of PI3K/AKT signaling pathways, suggesting ALT as a potential therapeutic candidate for osteosarcoma.

Elevated OPRD1 promoter methylation in Alzheimer's disease patients.

Aberrant DNA methylation has been observed in the patients with Alzheimer's disease (AD), a common neurodegenerative disorder in the elderly. OPRD1 encodes the delta opioid receptor, a member of the opioid family of G-protein-coupled receptors. In the current study, we compare the DNA methylation levels of OPRD1 promoter CpG sites (CpG1, CpG2, and CpG3) between 51 AD cases and 63 controls using the bisulfite pyrosequencing technology. Our results show that significantly higher CpG3 methylation is found in AD cases than controls. Significant associations are found between several biochemical parameters (including HDL-C and ALP) and CpG3 methylation. Subsequent luciferase reporter gene assay shows that DNA fragment containing the three OPRD1 promoter CpGs is able to regulate gene expression. In summary, our results suggest that OPRD1 promoter hypermethylation is associated with the risk of AD.