BACKGROUND The epithelial-mesenchymal transition (EMT) has been shown to be involved in the process of invasion and metastasis of prostate cancer. SIRT1 is the mammalian homologue of the silent information regulator 2 (Sir2) gene, and is abnormally expressed in prostate cancer cells. Therefore, it is hypothesized that SIRT1 mediates the invasion/metastatic ability of prostate cancer via EMT regulation. This study thus investigated the effect of SIRT1 gene on the invasion and migration of prostate cancer cell line PC-3 via the small interference RNA (siRNA) against SIRT1. MATERIAL AND METHODS SiRNA construct was transfected into PC-3 cells, which were tested for the cell migration and invasion ability by scratch assay and Transwell migration assay, respectively. Expression levels of vimentin, E-cadherin, and N-cadherin were further quantified by Western blotting and RT-PCR. RESULTS Both mRNA and protein levels of SIRT1 were depressed after siRNA transfection, along with weakened migration and invasion ability of PC-3 cells. Elevated E-cadherin and suppressed N-cadherin and vimentin were observed in those transfected cells. CONCLUSIONS The silencing of SIRT1 gene in PC-3 cells can suppress the movement, migration, and invasion functions of prostate cancer cells, possibly via the down-regulation of mesenchymal markers vimentin and N-cadherin accompanied with up-regulation of epithelial marker N-cadherin, thus reversing the EMT process.

This study aimed to determine the effects of long-term running exercise on spatial learning, spatial memory, and cortical capillaries in aged rats.

Lamivudine (LMV), as the preferred oral drug for use in treatment of HBV, always results in development of resistance mutations after long-term treatment. In this study we investigated chronic hepatitis B (CHB) patients in southern China to determine whether different HBV genotypes affect the incidence of LMV resistance mutations.

BACKGROUND Chronic spinal cord injury (CSCI) is a worldwide clinical problem. We aimed to reveal differentially expressed (DE) lncRNAs and to find associated pathways that may function as targets for CSCI therapy. MATERIAL AND METHODS After a CSCI rat model was confirmed by the Basso Beattie Bresnahan (BBB) scale and the Magnetic Resonance Imaging (MRI) test, microarray analysis was used to obtain the expression profile of DE lncRNAs between CSCI rats and corresponding control rats. Then, bioinformatics analyses, including GO and KEGG pathway analysis, DE lncRNAs-mRNAs co-expression analysis, and several databases, were used to examine the function of these DE lncRNAs. Finally, quantitative real-time PCR (qRT-PCR) was used to evaluate the expressions of the 5 most significantly changed lncRNAs, Col6a1, and miR-330-3p. RESULTS Our study identified 1266 DE lncRNAs and 847 DE mRNAs, among which lncRNA6032 was significant up-regulated. Furthermore, the expressions of miR-330-3p and Col6a1 associated with lncRNA6032 were down-regulated and up-regulated, respectively. CONCLUSIONS Our results showed that the abundance of DE lncRNAs may be associated with the risk of CSCI outcome and revealed a potential competitive endogenous RNA (ceRNA) pathway involved in CSCI. Further experiments in vivo and in vitro were essential to uncover the exact mechanism of this ceRNA pathway.

BACKGROUND Endothelial progenitor cells (EPCs) are regarded as promising targeted vectors for delivering therapeutic genes or agents in cancer therapy. The purpose of this study was to investigate the role of intravenously administered KAI1/CD82 genetically transduced EPCs in the tumorigenesis and metastasis of nasopharyngeal carcinoma (NPC). MATERIAL AND METHODS EPCs were isolated from human umbilical cord blood, expanded in culture, and stably transduced with lentiviral vectors expressing KAI1/CD82. The KAI1/CD82 EPCs were injected intravenously into nude mice bearing human NPC xenografts. Tumor growth and the incidence of liver and lung metastases were observed. Expression of KAI1/CD82 was determined by immunofluorescent staining. RESULTS The NPC model was successfully established. Tumor growth was not suppressed when mice were injected with KAI1/CD82 EPCs (KAI1/CD82 EPCs group) compared with when non-transduced EPCs was present (EPCs group) or the control (1.485±0.234, 1.388±0.204, and 1.487±0.223g, respectively; P>0.05). However, the incidence of lung metastasis was significantly reduced in the KAI1/CD82+ EPCs group compared with the EPCs group and the control group (10%, 55% and 45%, respectively; P=0.005), and there was a significant decrease in the number of metastatic foci on the lung surface (17.50±3.54, 34.27±5.35, and 38.44±9.63 respectively; P=0.007). Moreover, KAI1/CD82 was expressed in lung metastatic foci of the KAI1/CD82 EPCs group, but not in the EPCs group and control group. CONCLUSIONS EPCs can be used as a delivery vehicle for suppressor genes KAI1/CD82 to NPC, and the migration of KAI1/CD82 genetically engineered EPCs can inhibit NPC lung metastasis in a mouse model.

BACKGROUND Chronic myelogenous leukemia (CML) has unsatisfactory treatment efficacy at present. As the major component of red orpiment, tetra-arsenic tetra-sulfide (As4S4) has been recently used in treating leukemia, but with unclear mechanism targeting CML. MicroRNA (miR) is a group of endogenous non-coding RNAs regulating pathogenesis. MiR181 has been shown to exert important roles in tumor progression. The relationship between miR181 and As4S4 in inducing K562 cell apoptosis, however, is still unclear. MATERIAL AND METHODS CML cell line K562 was cultured in vitro in a control group and in groups receiving various dosages (20 μM and 40 μM) of As4S4. MTT assay was employed to detect the effect on K562 cell survival. MiR181 expression was quantified by real-time PCR. MTT assay and assay kit were used to determine K562 cell survival and caspase 3 expression. Cell apoptosis was assessed by flow cytometry. Bcl-2 expression was determined by real-time PCR and Western blotting. RESULTS As4S4 significantly suppressed proliferation of K562 cells (p<0.05) and decreased miR181 expression, and increased caspase3 activity compared to the control group. It can induce K562 cell apoptosis via remarkably down-regulating mRNA and protein expressions of Bcl-2 (p<0.05). CONCLUSIONS As4S4 can facilitate K562 cell apoptosis via down-regulating miR181, inhibiting Bcl02 expression, and enhancing apoptotic protein caspase3 activity.

BACKGROUND Bronchopulmonary dysplasia (BPD) is a major complication of extreme prematurity, characterized by alveolar simplification and pulmonary malfunction. Hyperoxia-induced lung injury in neonatal rats has been used as a model of BPD, as indicated by lung architectural change and alveolar simplification that resembles clinical feature of BPD. ß-defensin-2 (BD2) plays an important role in lung diseases by inhibiting inflammation response. However, little is known about its role in BPD. The aim of this study was to determine the effect of human BD2 (hBD2) gene on hyperoxia-induced animal model of BPD. MATERIAL AND METHODS The neonatal rats were exposed to 90% oxygen (O₂) continuously for 14 days to mimic the BPD-like lung injury. These rats were then randomly assigned to the following four groups: in room air (air), in 90% O₂, in 90% O₂ with null adenovirus vector infection (O₂+Ad), and in 90% O₂ with gene therapy through adenovirus transfected hBD2 (O₂+Ad-hBD2). Morphology of lungs, pulmonary function and expression of inflammatory cytokines on P7, P10, P14, and P21 were documented and compared across the 4 groups. RESULTS The overexpression of hBD2 mediated by the adenovirus vector was successfully constructed. hBD2 gene therapy increased hBD2 mRNA expression, increased radial alveolar count (RAC), lung volume and compliance, decreased mean linear intercept (MLI), tissue damping, and elastance. Furthermore, pro-inflammatory cytokines IL-1ß, IL-6, and TNF-alpha were inhibited and anti-inflammatory cytokines IL-10 was increased in the lungs of rats in O₂+Ad-hBD2 group. CONCLUSIONS In hyperoxia-induced rat models of BPD, hBD2 promotes alveolarization and improves pulmonary function. The mechanism may contribute in alleviating inflammation response and inhibiting pro-inflammatory factors including IL-1ß, IL-6, and TNF-alpha.

BACKGROUND Keratitis is a complex condition in humans and is the second most common cause of legal blindness worldwide. MATERIAL AND METHODS To reveal the genomic loci underlying keratitis, we performed functional annotations of SNP-based and gene-based genome-wide association studies of keratitis in the UK Biobank (UKB) cohort with 337 199 subjects of European ancestry. RESULTS The publicly available SNP-based association results showed a total of 34 SNPs, from 14 distinct loci, associated with keratitis in the UKB. Gene-based association analysis identified 2 significant genes: IQCF3 (p=2.0×10⁻⁶) and SOD3 (p=2.0×10⁻⁶). Thirty-two candidate genes were then prioritized using information from multiple sources. The overlap of IQCF3 in these 2 analyses resulted in a total of 33 hub genes. Functional annotation of hub genes was performed and transcriptional factors of IQCF3 and SOD3 were predicted. CONCLUSIONS A total of 34 SNPs from 14 distinct loci were identified as being associated with keratitis, and 32 candidate genes were then prioritized. In addition, IQCF3 and SOD3 were identified by their p values through gene-based tests on the basis of individual SNP-based tests. The functional relationship between these suspect genes and keratitis suggest that IQCF3 and SOD3 are candidate genes underlying keratitis.

BACKGROUND This retrospective study from a single center aimed to compare the safety and clinical outcomes of arthroscopic surgery vs open surgical repair of the anterior talofibular ligament (ATFL). MATERIAL AND METHODS We randomly divided 80 patients with ATFL injury divided into 2 groups: an open surgery group and an arthroscopic group. The operation time, intraoperative bleeding volume, and the postoperative recovery time of all patients were analyzed. The anterior displacement and talus tilt angle, the American Orthopedic Foot and Ankle Society Ankle-Hindfoot Score (AOFAS), the Jersey Shore Science Fair (JSSF) ankle-hindfoot scale score, and the Karlsson Ankle Functional Score (KAFS) were compared at 6 months, 1 year, and 2 years after surgery. We collected data on the incidence of postoperative complications during follow-up. All significant results were supported with a P value. RESULTS The operation time, intraoperative bleeding volume, and postoperative recovery time in the arthroscopic group were better than in the open group (P<0.05). The AOFAS, JSSF, and KAFS in the arthroscopic group were better than in the open group at 6 months after the operation (P<0.05). The AOFAS, JSSF, and KAFS scale scores were not significantly different between the 2 groups at 1 year and 2 years after the operation (P<0.05). CONCLUSIONS The findings from this retrospective study showed that the use of arthroscopic surgical repair of the ATFL is a safe minimally invasive technique with reduced blood loss and surgical duration and good clinical outcomes.

BACKGROUND The network pharmacological approach was used to identity the anti-colorectal cancer (CRC) targets of formononetin (FN) and the molecular mechanisms of FN against CRC. MATERIAL AND METHODS A tool of the DisGeNET database was used for collection of CRC-based targets. Other tools of SuperPred, herbal ingredients target (HIT), and SwissTargetPrediction databases were applied in prediction of pharmacological targets of FN against cancer. A protein-protein interaction (PPI) network of FN against CRC was obtained by using a STRING database. All top biological functional processes and signaling pathways of FN against CRC were identified by using Database for Annotation, Visualization and Integrated Discovery (DAVID) software and Omicshare cloud platform. RESULTS The most key anti-CRC targets of FN were identified as tumor protein p53 (TP53), cytochrome P450 3A4 (CYP3A4), ATP binding cassette subfamily G member 2 (ABCG2), tumor necrosis factor (TNF), epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 2 (ERBB2), and cytochrome P450 1A1 (CYP1A1). In further assays, the treatment of CRC by FN was mainly involved in biological functional processes of reactive oxygen species metabolic process, positive regulation of transcription, DNA-templated, positive regulation of nucleic acid-templated transcription, and positive regulation of RNA metabolic process. anti-CRC by FN of signaling pathways were associated with amyotrophic lateral sclerosis (ALS), allograft rejection, cytokine-cytokine receptor interaction, asthma, mitogen-activated protein kinase (MAPK) signaling pathways, and others. CONCLUSIONS The anti-CRC molecular mechanisms of FN are implicated in suppression of cellular proliferation and regulation of cancer-related metabolic pathways. Interestingly, 8 optimal biological targets may be used as potential molecular markers for predicting and treating CRC.

BACKGROUND The aim of this study was to detect the expression of fork-head box D3 (FOXD3) and investigate its diagnostic value in patients with non-small cell lung cancer (NSCLC). MATERIAL AND METHODS The relative expression of FOXD3 at mRNA and protein levels was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting analysis, respectively. Chi-square test was used to explore the relevance of FOXD3 expression with clinical features of NSCLC patients. A receiver operating characteristic (ROC) curve was built to estimate the diagnostic value of FOXD3 in distinguishing NSCLC patients from healthy controls. RESULTS Serum FOXD3 expression was weakly expressed in NSCLC patients compared to the controls at mRNA and protein levels (P<0.001) and low FOXD3 expression was positively correlated with TNM stage, lymph node metastasis, and differentiation. The ROC curve indicated that FOXD3 acts as a diagnostic bio-marker for NSCLC patients, with an AUC of 0.826 corresponding to a sensitivity of 77.1% and a specificity of 74.6%, and an optimal cutoff point of 2.38. CONCLUSIONS Decreased expression of serum FOXD3 was observed in NSCLC patients, and it was found to be a potential molecular marker for the diagnosis of NSCLC.

BACKGROUND Pediatric liver transplantation is used to treat children with end-stage liver disease. This study explored the research hotspots and bibliometric characteristics of pediatric liver transplantation through a variety of bibliometric analysis software. We conducted hotspot analysis to help determine important directions for future scientific research. MATERIAL AND METHODS The study samples were articles related to pediatric liver transplantation published in PubMed in the past 5 years. The high-frequency keywords are extracted by BICOMB software, and then a binary matrix and a common word matrix were constructed. Gcluto software was used to perform double-clustering and visual analysis on high-frequency words, and then we obtained hot area classification. Strategic coordinates are constructed using Excel. Citespace and VOSviewer software are used for further analysis and bibliometric data visualization. RESULTS A total of 36 high-frequency words were found in the 4118 studies. A peak map was drawn through double-cluster analysis. Biclustering analysis was used to calculate the concentricity and density of each hotspot. We obtained the top 10 countries/regions engaged in pediatric liver transplantation research. VOSviewer was used to visualize the co-author map. CONCLUSIONS We found 5 clusters and 7 aspects for pediatric liver transplantation. Additionally, calculation results showed that post-transplant lymphoproliferative disorder in pediatric patients and outcomes of multivisceral transplantation seem very promising. This conclusion is of great value for future exploratory research.

BACKGROUND Placenta accreta spectrum (PAS) includes placenta increta, placenta percreta, and placenta accreta. PAS is due to abnormal decidualization and can lead to severe maternal hemorrhage and occurs in up to 3% of women with central placental previa (CPP). This study from a single center aimed to compare the magnetic resonance imaging (MRI) changes in the lower uterine segment in pregnant women with CPP, with and without PAS. MATERIAL AND METHODS This retrospective study includes 90 pregnant women with PAS and 66 pregnant women without PAS. All participants were confirmed to have CPP by MRI. Eight MRI parameters were assessed and compared with perinatal outcomes for mothers and newborns. RESULTS The pregnancies in the non-PAS group had less operative time (P=0.001), less intrapartum hemorrhage (P<0.001), and less blood transfusion than the PAS group (P<0.001). The 8 MRI variables with different odds ratios were placenta thickness (4.20), cervical lengths (3.27), placental dark T2 bands area (5.10), cervical marginal sinus (3.04), bladder bulge (3.55), myometrial thinning (6.41), lower uterine segment bulge (4.61), and placental signals in the cervix (9.14). The sensitivity and specificity of MRI in the diagnosis of PAS were 82.22% and 91.09%, respectively, by the combined 8 MRI features, and the area under the curve (AUC) was 0.816. CONCLUSIONS The findings from this study showed that MRI of the lower uterine segment had high sensitivity and specificity for the diagnosis of PAS in pregnant women with CPP.

BACKGROUND Osteosarcoma (OS), an aggressive malignant neoplasm, is the most common primary bone cancer mainly in adolescents and young adults. Differentially expressed modules tend to distinguish differences integrally. Identifying modules individually has been crucial for understanding OS mechanisms and applications of custom therapeutic decisions in the future. MATERIAL AND METHODS Samples came from individuals were used from control group (n=15) and OS group (n=84). Based on clique-merging, module-identification algorithm was used to identify modules from OS PPI networks. A novel approach - the individualized module aberrance score (iMAS) was performed to distinguish differences, making special use of accumulated normal samples (ANS). We performed biological process ontology to classify functionally modules. Then Support Vector Machine (SVM) was used to test distribution results of normal and OS group with screened modules. RESULTS We identified 83 modules containing 2084 genes from PPI network in which 61 modules were significantly different. Cluster analysis of OS using the iMAS method identified 5 modules clusters. Specificity=1.00 and Sensitivity=1.00 proved the distribution outcomes of screened modules were mainly consistent with that of total data, which suggested the efficiency of 61 modules. CONCLUSIONS We conclude that a novel pipeline that identified the dysregulated modules in individuals of OS. The constructed process is expected to aid in personalized health care, which may present fruitful strategies for medical therapy.

BACKGROUND Bladder cancer is a major widespread tumor of the genitourinary tract. Around 30% of patients with superficial cancers develop invasive and metastatic pathology. MATERIAL AND METHODS Some new heterocyclic 4-methyl coumarin derivatives were designed using molecular modeling studies to evaluate their potential against bladder cancer lines T24 and RT-4. The designed compounds that showed good binding affinity to T24 and RT4 were synthesized, with excellent yield. The synthesized compounds after structural evaluation were further evaluated for their antiproliferative activity by cell viability assay, cell cycle analysis, and apoptosis assay. RESULTS The compound BC-14 exhibited the best cytotoxicity against T24 cells, but were not highly active against RT4 cells. CONCLUSIONS The results of the present study may suggest the selectivity pattern of the synthesized compounds. These results should be explored further with chemical modification for other cancer types.

Non-muscle-invasive urothelial carcinoma of the bladder has a high rate of recurrence, necessitating use of a variety of adjuvant treatments. A single, immediate post-operative administration of chemotherapy is an important measure for reducing the rate of recurrences, but the overall effect is not satisfactory and the treatment is a burden to the patient. Hence, there is a need for a new, more effective and cheaper agent for adjuvant treatment of non-muscle-invasive bladder carcinomas. Although cationic surfactants such as benzalkonium salts are used clinically and hygienically for the control of bacterial growth, there have been reports that cationic surfactants such as benzethonium chloride induce apoptosis in normal and in cancerous cells. We report our experience with benzalkonium bromide (BB) accidentally administered into a patient's bladder as saline. It caused severe hematuria and pain, but after a week of persistent administration in the bladder, the patient was cured, as supported by evidence from cystoscopy, indicating that BB destroys bladder mucosa and suggesting that BB maybe a novel agent for use in a single, immediate post-operative administration. We present preliminary data to support this hypothesis and provide discussion we hope will be useful as the foundation for further experiments.

BACKGROUND Collagen type VI alpha 3 chain (COL6A3) has been proven to be a biomarker in the occurrence and development of bladder cancer, which is the most common malignant tumor in the urinary system. This study aimed to explore the effect and molecular mechanism of COL6A3 on EMT in vitro induced by TGF-β/Smad in bladder carcinoma. MATERIAL AND METHODS There were 42 patients included in the Kaplan-Meier survival analysis. A cell counting kit-8 (CCK-8) assay and an angiogenesis assay were used to measure cell proliferation and tube formation, respectively. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) were conducted for the proteins and mRNAs expression. RESULTS COL6A3 was highly expressed in tissues and cells of bladder cancer. COL6A3 silencing could inhibit the cell proliferation and angiopoiesis. In addition, COL6A3 silencing obviously suppressed the levels of matrix metalloproteinase-2 (MMP2), Matrix metalloproteinase-9 (MMP9), and vimentin. On the contrary, the levels of epithelium-specific cell-cell adhesion molecule (E-cadherin) and tumor inhibitor of metalloproteinase-1 (TIMP-1) were significantly increased. Furthermore, we found that COL6A3 silencing reduced the activity of p-Smad2, p-Smad3, and transforming growth factor β (TGF-β). CONCLUSIONS COL6A3 could influence the viability and angiogenesis of bladder cancer cells. COL6A3 may have a certain relationship with the TGF-β/Smad-induced EMT process.

BACKGROUND Chlamydiae are spread globally and cause infectious diseases in both humans and animals. The existing detection methods for this disease have numerous shortcomings, including low sensitivity, time consuming procedures, and high contamination vulnerability. MATERIAL AND METHODS To overcome shortcomings for detecting animal chlamydiosis, a multiplex quantitative polymerase chain reaction (PCR) assay was established for simultaneously detecting and differentiating 3 Chlamydia species (C. pecorum, C. abortus, and C. psittaci) by real time PCR based on TaqMan-MGB technology. RESULTS The limit of detection was 20.2 copies/µL for Chlamydophila (Cp.) abortus, 30.8 copies/µL for Cp. pecorum, and 16 copies/µL for Cp. psittaci. This method has good repeatability and stability as coefficients of variation range from 0.04% to 1.38%. Furthermore, compared with OIE (World Organization for Animal Health) recommended PCR assay and previously reported animal chlamydia shell PCR, this multiplex PCR assay demonstrated 99% concordance in detecting clinical samples of porcine nasal swabs and vaginal swabs. CONCLUSIONS The novel established method in this study was able to detect 3 types of Chlamydia species simultaneously, and had high sensitivity, strong specificity, and good stability. It provided a rapid, reliable, and convenient method for epidemiological and clinical diagnosis of chlamydiosis in animals.

BACKGROUND Measles morbidity and mortality were significantly reduced after the measles vaccine was introduced in China in 1965. However, measles outbreaks easily occur in densely populated areas, especially where there is no universal vaccination. The outbreak that occurred in Shenzhen, the Chinese city with the largest internal immigration, provides a lesson in measles virus mutation and measles prevention. The present study is a phylogenetic analysis of measles viruses and comparison of clinical signs between individuals with and without vaccination. MATERIAL AND METHODS We performed phylogenetic analysis of the nucleoprotein (N) genes of measles virus from 129 measles patients in Shenzhen from January 2015 to July 2019. Phylogenetic trees were constructed using the neighbor-joining method. RESULTS The phylogenetic analysis showed all viruses were classified into genotype H1. In addition, there is often a seasonal measles outbreak in July each year. The clinical data showed that patients who were unvaccinated were more likely to have coughing, chronic bronchitis, conjunctivitis, catarrh, Koplik spots, and diarrhea. Children of migrant workers and those living in mountainous and rural districts accounted for most measles cases. CONCLUSIONS Our results showed there was a seasonal measles outbreak in Shenzhen Children's Hospital. All the measles virus from 129 measles patients were H1 viruses. The clinical signs also showed a difference between unvaccinated and vaccinated patients. Moreover, most of the unvaccinated patients came from migrant worker families. We suggest there is a need for increased health promotion and vaccination programs for migrant workers and people living in remote villages.

BACKGROUND Specific T cell phenotype has been reported to potentially contribute to the development of angiotensin II (Ang II)-induced several vascular disorders. Type 2 diabetes mellitus (T2DM) is intimately associated with cardiovascular disease. The present study aimed to investigate the relationship between T cell phenotypes and Ang II in T2DM patients combined with carotid atherosclerosis (CA). MATERIAL AND METHODS This study was performed on 50 patients with T2DM in our hospital. Based on the presence of CA, they were divided into CA group (presence of CA, n=30) or T2DM group (absence of CA, n=20). Additionally, 10 healthy participants were selected as controls. Basic characteristics of all participants were collected and recorded. Peripheral blood mononuclear cells (PBMCs) isolated from patients and controls with or without Ang II and Ang II receptor blocker (ARB) treatment were used to detect Th1, Th2, and Th17 cell proportions, mRNA levels of T-bet, GATA3, and RORγt as well as the expression of IFN-γ, IL-4, and IL-17 by flow cytometry, ELISA, and Real-Time PCR. RESULTS Ang II levels were notably higher in patients in the CA group than those in the T2DM and control group (p<0.05). Th1 and Th17 positive cells, mRNA levels of T-bet and RORgt as well as the expression of IFN-γ and IL-17 were significantly increased in the CA group compared with the T2DM group and control group (p<0.05). Moreover, the activities of T cells and related cytokines were significantly increased of healthy controls after Ang II treatment (p<0.05), while these changes were notably weakened by ARB treatment (p<0.05). CONCLUSIONS Ang II promotes the development of CA in T2DM patients by regulating T cells activities.