Fuzi-Lizhong Decoction Alleviates Nonalcoholic Fatty Liver Disease by Blocking TLR4/MyD88/TRAF6 Signaling.

Background: Fuzi-Lizhong decoction (FLD) derives from the ancient Chinese Pharmacopoeia and has been clinically used for years. The present study aimed to investigate the activities and underlying mechanisms of FLD against nonalcoholic fatty liver disease (NAFLD). Methods: In vivo studies were conducted by inducing NAFLD in rats with a high-fat diet, and in vitro studies were performed on HL-7702 cells treated with oleic and linoleic acids. Total cholesterol (TC), triglyceride (TG), and blood glucose (Glu) levels were detected using an automatic biochemical analyzer. The expression of IL-2, IL-6, and TNF-α in sera and cell culture supernatants was measured by ELISA. The mRNA and protein levels of TLR4, MyD88, and TRAF6 were measured in liver tissue and HL-7702 cells using reverse transcription-quantitative polymerase chain reaction and western blot. Results: FLD significantly reduced the TC, TG, Glu, FFA, IL-2, IL-6, and TNF-α levels in NAFLD rats and HL-7702 cells. Analysis of liver lipid content by Oil Red O staining revealed a significant increase in hepatic lipid accumulation in rats with NAFLD, but this lipid accumulation was reversed by FLD treatment. In addition, the mRNA expression levels of TLR4, MyD88, TRAF6, and NF-κB p65 as well as the protein levels of TLR4, MyD88, TRAF6, and NF-κB p65 were decreased after FLD treatment. FLD significantly reduced inflammation and improved collagen accumulation in vivo and in vitro by inhibiting the activation of the TLR4/MyD88/TRAF6 signaling pathway. Conclusions: FLD exerted potent protective effects against NAFLD via TLR4/MyD88/TRAF6 signaling. These findings provide novel insights into the mechanisms whereby this compound acts as an anti-inflammatory agent and highlight the potential application of FLD in the treatment of acute liver failure (ALF).

Curcumin protects against manganese-induced neurotoxicity in rat by regulating oxidative stress-related gene expression via H3K27 acetylation.

Long-term manganese exposure causes a neurodegenerative disorder referred to as manganese poisoning, but the mechanism remains unclear and no specific treatment is available. Oxidative stress is widely recognised as one of the main causes of manganese-induced neurotoxicity. In recent years, the role of histone acetylation in neurodegenerative diseases has been widely concerned. curcumin is a natural polyphenol compound extracted from the rhizome of turmeric and exhibits both antioxidant and neuroprotective properties. Therefore, we aimed to investigate whether and how curcumin protects against manganese-induced neurotoxicity from the perspective of histone acetylation, based on the reversibility of histone acetylation modification. In this study, rats were treated with or without curcumin and subjected to long-term manganese exposure. Results that treatment of manganese decreased the protein expression of H3K18 acetylation and H3K27 acetylation at the promoters of oxidative stress-related genes and inhibited the expression of these genes. Nevertheless, curcumin increased the H3K27 acetylation level at the manganese superoxide dismutase (SOD2) gene promoter and promoted the expression of SOD2 gene. Oxidative damage in the rat striatum as well as learning and memory dysfunction were ameliorated after curcumin treatment. Taken together, our results suggest that the regulation of oxidative stress by histone acetylation may be a key mechanism of manganese-induced neurotoxicity. In addition, curcumin ameliorates Mn-induced neurotoxicity may be due to alleviation of oxidative damage mediated by increased activation of H3K27 acetylation at the SOD2 gene promoter.

Xiaoyao Powder in the treatment of non-alcoholic fatty liver disease: A systematic review and meta-analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide with alarming prevalence. Due to its complex pathogeneses and considerable individual heterogeneity in disease, there is no specific medication to NAFLD safely and effectively. Therefore, there is a great need to explore complementary and alternative therapies. Xiaoyao Powder (XYP), a classic Chinese formula, has been tremendously applied to gastrointestinal diseases, especially non-alcoholic fatty liver disease. However, the efficacy and safety of XYP have not been fully assessed. AIM OF THE STUDY: To assess the effectiveness and safety of XYP for NAFLD. MATERIALS AND METHODS: The assigned registration number on the PROSPERO platform of this meta-analysis is CRD42020192154, and we strictly followed the protocol. We searched eight primary databases from their inception to June 2020. Two authors independently identified random controlled trials (RCTs) of XYP for NAFLD and evaluated the quality of the retrieved articles by Cochrane accessing risk bias tool. At least one of the following indices was thoroughly documented for outcome measurement: total effective rate, total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamyl transpeptidase (GGT), body mass index (BMI), and adiponectin. We calculated risk ratio (RR) and mean difference (MD) for dichotomous data and continuous variables with a 95% confidence interval (CI). R 4.0.5 software was employed for data synthesis. RESULTS: Consequently, we identified 12 studies with 1012 participants. XYP, whether individually or combined with essential treatment, ameliorated NAFLD regardless of the course of the disease or curative duration. This benefit was mainly driven by regulating levels of serum markers, involving TC, TG, ALT, AST, GGT, and adiponectin. Three studies where statins were concerned about drug safety reported several adverse events with clinical symptoms, varying from flatulence, constipation, and diarrhea to rash, whereas others did not. CONCLUSION: Our findings provided evidence that XYP is a therapeutic option to treat NAFLD effectively and safely. Notwithstanding, a precise and comprehensive conclusion calls for RCTs on a larger scale with more rigorous designs considering the inferior methodological quality and limited retrieved articles.

[Research progress on mechanisms of traditional Chinese medicine in prevention and treatment of postoperative peritoneal adhesion].

Peritoneal adhesion is one of the common complications after abdominal operation, which could seriously affect the quality of life in patients. Although the development of modern surgical technology and the improvement of doctors' operation level have reduced the incidence of peritoneal adhesion to a certain extent, due to the lack of special treatment drugs, the therapeutic effect still cannot meet the expectations and requirements of clinicians and patients. Traditional Chinese medicines(TCM) have unique advantages and remarkable curative effect in the treatment of peritoneal adhesion, and they can play an important role in regulating multiple pathological links. However, the relevant researches and product development of TCM against peritoneal adhesion have not attracted enough attention from industry scholars. As for the related work that has been carried out, most of the studies on the efficacy and mechanism are not thorough and systematic enough, seriously restricting the industrial development in this field. In this paper, the efficacy and mechanism were systematically described and summarized based on the review of papers in the recent years, so as to provide a reference for the thorough study of TCM in the prevention and treatment of peritoneal adhesions, and promote the deep development and industrialization process of related products.

Protective Effect of Fuzi Lizhong Decoction against Non-alcoholic Fatty Liver Disease via Anti-inflammatory Response through Regulating p53 and PPARG Signaling.

Fuzi Lizhong decoction (FLD) is derived from an ancient Chinese Pharmacopoeia and has been used in clinical treatment for years. The present study aimed to investigate the activities and underlying mechanisms of FLD against non-alcoholic fatty liver disease (NAFLD). Network pharmacology analysis demonstrated that FLD might affect NAFLD through regulating p53 and peroxisome proliferator activated receptor gamma (PPARG), which has been confirmed in vitro and in vivo. In vivo NAFLD was induced in rats by a high-fat diet, and in vitro studies were performed on HL-7702 cells treated with oleic acid and linoleic acid. We showed that FLD significantly improved NAFLD by regulating the immune system to induce the release of interleukin-10 (IL-10), interferon-α (IFN-α), and IFN-ß through activating p53 signaling and inhibiting PPARG signaling in vivo and in vitro. P53 inhibition induced by NAFLD was recused by FLD, while PPARG overexpression induced by NAFLD was inhibited by FLD. In addition, NAFLD resulted in increased levels of total cholesterol, triglyceride, and blood glucose in the serum and free fatty acid in the liver, which were reduced by FLD treatment. Evidently, FLD exhibited potent protective effects against NAFLD via p53 and PPARG signaling. Our study could provide novel insights into the mechanisms of FLD as an anti-inflammatory candidate for the treatment of NAFLD in the future.

Histone demethylase JHDM2A regulates H3K9 dimethylation in response to arsenic-induced DNA damage and repair in normal human liver cells.

Long-term arsenic exposure is a worldwide public health problem that causes serious harm to human health. The liver is the main target organ of arsenic toxicity; arsenic induces disruption of the DNA damage repair pathway, but its mechanisms remain unclear. In recent years, studies have found that epigenetic mechanisms play an important role in arsenic-induced lesions. In this study, we conducted experiments in vitro using normal human liver cells (L-02) to explore the mechanism by which the histone demethylase JHDM2A regulates H3K9 dimethylation (me2) in response to arsenic-induced DNA damage. Our results indicated that arsenic exposure upregulated the expression of JHDM2A, downregulated global H3K9me2 modification levels, increased the H3K9me2 levels at the promoters of base excision repair (BER) genes (N-methylpurine-DNA glycosylase [MPG], XRCC1 and poly(ADP-ribose)polymerase 1) and inhibited their expression levels, causing DNA damage in cells. In addition, we studied the effects of overexpression and inhibition of JHDM2A and found that JHDM2A can participate in the molecular mechanism of arsenic-induced DNA damage via the BER pathway, which may not be involved in the BER process because H3K9me2 levels at the promoter region of the BER genes were unchanged following JHDM2A interference. These results suggest a potential mechanism by which JHDM2A can regulate the MPG and XRCC1 genes in the process of responding to DNA damage induced by arsenic exposure and can participate in the process of DNA damage repair, which provides a scientific basis for understanding the epigenetic mechanisms and treatments for endemic arsenic poisoning.

[Analysis of Causes and Condition of Blood Donation Reaction Under Collective Emergency Blood Donation].

OBJECTIVE: To explore the causes and specific conditions of blood donation reaction under the collective emergency unpaid blood donation, and to provide theoretical basis and decision-making reference for drafting the collective emergency unpaid blood donation and blood donation safety. METHODS: Through a combination of prospective and retrospective models, and statistical methods were used to analyze the causes and conditions of the blood donation response of 10401 people participating in collective emergency unpaid blood donation during 2016.1-2018.8. RESULTS: A total of 10401 person-times donated blood in a sitting manner, and a total of 293 blood donation reactions occurred. By improving the blood donation services year by year, the moderate blood donation reaction during the year 2017 and 2018 was significantly lower than that in 2016 (P＜0.05). In the actual blood donation group of≤100, 200, 300 and 400 ml, the incidence of blood donation reaction was statistically significant (P＜0.05); the incidence of blood donation reaction in the blood donors for 1,2,3 and ＞3 drnations was also statistically significant (P＜0.05); the blood donation reactions rate of B antigen containers was significantly different from the donors without B antigen (P＜0.05); the incidence of blood donation reaction with related to the weight of the donor. CONCLUSION: The blood donation reaction of collective emergency unpaid blood donation closely relates with mental factors, blood donation service, blood donation frequency and body weight of the blood donor. The first blood donation is more likely to produce blood donation reaction. The blood donation volum≤ 100 ml from blood donors is resulted mostly from blood donation reactions.

Characterization of Sample Loss Caused by Competitive Adsorption of Proteins in Vials Using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.

Sample loss caused by competitive protein adsorption on solid surfaces from complex samples remains to be a major hurdle in sensitive analyses of proteins. No label-free techniques can easily quantify individual proteins adsorbed on irregular surfaces of Eppendorf vials or Falcon tubes, which are commonly used to contain complex biological samples. Multiplexed characterization of such adsorption by different proteins is technically challenging. Herein, we developed a direct protein analysis based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the characterization of sample loss occurred on the curved surface with limited area. Using this simple and easily accessible method, we discovered the effect of ethylenediaminetetraacetic acid on surface adsorption of different milk proteins, specifically an augmented loss of milk proteins in low-binding sample vials. In this study, we also identified severe biases of silver staining and established proteomics-based mapping of protein distribution in biological samples for absolute quantification of competitive protein adsorption on irregular surfaces.

Correction to: Evaluation of 22G fine-needle aspiration (FNA) versus fine-needle biopsy (FNB) for endoscopic ultrasound-guided sampling of pancreatic lesions: a prospective comparison study.

The HTML version of this article was updated to indicate that the copyright is with The Author(s).

Evaluation of 22G fine-needle aspiration (FNA) versus fine-needle biopsy (FNB) for endoscopic ultrasound-guided sampling of pancreatic lesions: a prospective comparison study.

BACKGROUND: To compare the diagnostic yield and safety of 22G endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) in the diagnosis of pancreatic solid lesions. METHODS: Between April 2014 and September 2015, 36 patients with pancreatic solid lesions were included for endoscopic ultrasound test. Patients were randomly divided into two groups: EUS-FNA (n = 18) and EUS-FNB (n = 18). Each nidus was punctured three times (15 ~ 20 insertions for each puncture) with a 22G needle. The core specimens were analyzed, and the diagnostic yields of FNA and FNB were evaluated. RESULTS: The procedure success rate was 100% with no complications. Cytological and histological examinations found that the diagnostic yield of FNB and FNA were both 83.3%. To get a definitive diagnosis, FNB needed fewer punctures than FNA (1.11 vs. 1.83; P  <  0.05). CONCLUSIONS: 22G EUS-FNB is a safe and effective way to diagnose pancreatic solid lesions. FNB required a lower number of needle passes to achieve a diagnosis compared with FNA.

Percutaneous transhepatic techniques for retrieving fractured and intrahepatically dislodged percutaneous transhepatic biliary drainage catheters.

Dislodged intrabiliary drainage devices, including catheters, endoprostheses, and stents, may further impair drainage and cause various local reactions, vascular and gastrointestinal tract complications. Endoscopic approaches for management of plastic biliary endoprostheses have been extensively discussed. However, in rare cases of fracture of percutaneous transhepatic biliary drainage (PTBD) catheters, only a percutaneous transhepatic technique for retrieving should be applied to avoid further damage by its rigid fragment. We present the adjusted techniques using either a goose neck snare, over-the-wire balloon catheter, or biopsy forceps with image demonstration and reviews. We encountered two patients with PTBD tube fracture and intrahepatic dislodgment. In both patients, percutaneous approaches were used for successfully retrieving and removing the fractured catheter through transhepatic tract: one with the use of a biopsy forceps, another with an inflatable balloon catheter.

Application of clips assisted with foreign body forceps in defect closure after endoscopic full-thickness resection.

BACKGROUND: This study was designed to evaluate the feasibility and efficacy of metallic clips assisted with foreign body forceps closing the gastric wall defect after endoscopic full-thickness resection (EFR) for gastric submucosal tumors (SMTs). METHODS: Eighteen patients with gastric SMTs originated from the muscularis propria were treated by EFR between September 2012 and June 2014. Twelve patients underwent endoscopic closure of the gastric wall defects after EFR with endoloop and metallic clips (endoloop string suture method, ESSM), and six patients with clips and foreign body forceps (clips assisted with foreign body forceps clip method, CFCM). RESULTS: No significant differences existed between the two groups in terms of demographics, clinical characteristics, and the size of the gastric wall defects. The average time spent in closing the gastric wall defects (14.83 ± 1.94 min for the CFCM group and 22.42 ± 5.73 min for the ESSM group) and hospitalization fees of the CFCM group were significantly lower than those of the ESSM group. The average hospitalization time of the two groups had no statistical significance. No single case had surgical intervention or complications, such as gastric bleeding, perforation, peritonitis, or abdominal abscess. CONCLUSION: The CFCM and the ESSM are safe and effective techniques for gastric defect closure after EFR for gastric SMTs. Because of the "chopsticks effect," the CFCM more suitable for the lesions located at the gastric fundus, the greater curvature or anterior wall of the gastric body and gastric antrum.

Delineation gross tumor volume based on positron emission tomography images by a numerical approximation method.

OBJECTIVE: A scheme, named SUV_Shape, for the gross tumor volume (GTV) delineation on positron emission tomography (PET) images was designed by a numerical approximation method, and evaluated during this study. METHODS: Twenty-one vacuous plastic balls of different shapes and sizes, their volumes ranged from 0.56 to 179.50 mL and were confirmed by a BL610 balance (Sartorius, Canada), consisted of four group models. Every group model was filled with a specific activity [18F]-FDG solution (55.1, 38.2, 23.7, and 36.3 kBq/mL) represented tumor, and fixed at the bottom of a barrel which was filled with unlike [18F]-FDG solution (5.4, 6.8, 8.1, and 4.0 kBq/mL, correspondingly) represented the background. The PET data of them were acquired by two-dimensional and three-dimensional mode in a PET/CT scanner (Discovery ST8, GE Healthcare, USA). The volume of each ball was measured by SUV_Shape, and the BL610 balance, labeled as GTVs and GTVt, respectively. Five rabbits implanted VX2 squamous carcinomas were acquired by [18F]-FDG PET/CT. These rabbits were mercy killed within 24 h after PET/CT acquisition. VX2 tumors were surgically removed, and their volumes were measured by SUV_Shape, and caliper, labeled as GTVs and GTVt. The Spearman's ρ between GTVs and GTVt were done. RESULTS: The tumor-background ratios in four groups of phantom models were 10.3, 5.6, 2.9, and 9.0, respectively. The relationship between GTVt and GTVs for phantom models was significant (Spearman's ρ > 0.95, P < 0.01), regardless of the different acquisition modes. Twelve VX2 tumor nodes or masses were measured; their GTVt ranged from 0.11 to 29.26 mL. The relationship between GTVt and GTVs was significant (Spearman's ρ = 0.893, P < 0.01) for animal tumor models. CONCLUSIONS: The SUV_Shape scheme could delineate tumors based on their radiopharmaceutical-avid PET images.

Expression and significance of P53 protein and MDM-2 protein in human gliomas.

BACKGROUND: P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed. METHODS: The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade I - II group: 5 cases of grade I, 119 cases of grade II; and grade III--IV group: 53 cases of grade III, and 35 cases of grade IV). RESULTS: The P53 positive rate was significantly higher in the glioma groups than in the control group (P < 0.0001). The P53 positive rate was significantly higher in glioma tissues of grade III - IV than in glioma tissues of grade I - II group (P = 0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P < 0.0001). There was no significant difference in the MDM-2 positive rate between the two glioma groups (P = 0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P = 0.069) CONCLUSIONS: Overexpression of P53 protein might be related to the occurrence and progression of glioma. Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.

[Clinicopathologic evaluation of 36 cases of allergic fungal sinusitis].

OBJECTIVE: To characterize clinicopathological features of allergic fungal sinusitis (AFS). METHODS: Thirty-six cases of AFS were retrieved from the department archival files of Beijing Tongren Hospital from 2002 to 2006. AB-PAS, GMS and MUC5B stain were performed using paraffin-embedded tissues of the cases. Ten cases with available fresh diagnostic tissue were investigated by electron microscopy. RESULTS: Patients included 21 males and 15 females. The age of patients ranged from 11 to 53 years. Atopy was very common in these patients. On plain CT scans, the affected nasal sinuses were filled with soft tissue shadow with patchy hyperdensity. The bony sinus wall showed areas of pressure erosion. Skin antigen tests showed fungal positivity in 31 of 36 cases. Serum levels of the total IgE and/or the specific fungal IgE were elevated in 20 cases. The eosinophil quantity was elevated in 23 cases. Fungal culture was positive in 10 cases. Gross examination showed thick putty secretions within the lesions. Light microscopy showed typical "eosinophilic mucin". Fungal elements were seen with AB-PAS, GMS and MUC5B stains. Electron microscopy demonstrated degranulation by the eosinophils. CONCLUSIONS: "Eosinophilic mucin" is the typical histopathological feature of AFS. AB-PAS, GMS and MUC5B staining methods can used to detect fungal species in mucin. Accurate diagnosis of AFS requires correlations among clinical findings, radiologic examinations, laboratory tests and histopathologic features. However, the ultimate diagnosis requires a histopathologic confirmation.

Increased expression of fascin associated with malignant transformation of sinonasal inverted papilloma.

BACKGROUND: Fascin, an actin binding protein, usually expressed at a low level in normal epithelium, but is significantly increased in transformed epithelial cells and several common carcinomas. In this study, we examined the expression of fascin by immunohistochemistry in sinonasal epithelium with chronic inflammation (control group), exophytic papilloma (EP), inverted papilloma (IP) with dysplasia and cancerated IP (including carcinoma in situ and invasive squamous cell carcinoma, SCC), and furthermore investigated the relationship between fascin expression and formation of malignant IP. METHODS: Fascin expression was immunohistochemically detected using monoclonal antibody against fascin in 86 paraffin embedded tissues, including 10 cases of sinonasal mucosa with chronic inflammation, 10 of EP, 45 of IP with dysplasia (45 cases were divided into three groups: IP with mild dysplasia, IP with moderate dysplasia, and IP with severe dysplasia, 15 cases each), and 21 of cancerated IP. RESULTS: The level of fascin expression was significantly higher in the neoplastic tissue than that in control group. Fascin expression increased gradually with the progression from sinonasal epithelium with chronic inflammation, IP with mild dysplasia, IP with moderate dysplasia, IP with severe dysplasia, to cancerated IP, and significant difference of fascin expression was observed between any two groups of the five. CONCLUSION: Precancerous lesions of IP exhibit elevated levels of fascin that may be associated with carcinogenesis of IP. Fascin may play a role in the formation of IP and EP.

Effect of chelating functional polymer on the size of CdS nanocluster formation.

Chelating poly(acrylates-co-2-methylacrylic acid 3-(bis-carboxymethylamino)-2-hydroxy-propyl ester) microspheres of diameter 250-310 nm were prepared by the soap-free emulsion polymerization method for varying amounts of GMA-IDA. Then CdS/copolymer composite was generated by chemical deposition on the surface of the copolymer microspheres. By XRD analysis it is found that the chelated CdS nanoparticles are a pure cubic zinc blende structure. The CdS/copolymer composite is examined by UV-vis absorbance, photoluminescence, and TEM observation. Average CdS nanoparticle size calculated from Henglein's empirical curve is in the range of 3.0-8.0 nm and varies according to the GMA-IDA molar ratio during polymerization, pH value during chelation, and postchelation annealing temperature. Higher ratio of chelating group, pH value, and annealing temperature produce larger CdS nanoparticles. As GMA-IDA ratio increases, photoluminescence exhibits a red shift from 510 to 520 nm, photoluminescence increases, and bandwidth decreases. Photoluminescence of the CdS nanoparticle becomes negligible when diameter exceeds 5 nm.

[The change of circulating endothelial cell in blood of rats and pulmonary small artery vascular endothelial cell under acute hypoxia].

AIM: To investigate changes of VEC and CEC under acute hypoxia. METHODS: Observe CEC in blood under acute hypoxia morphologically and count the number of CEC by optical microscope, measure LDH activity of young CEC and VEC by histochemical staining image analysis. RESULTS: LDH activities of VEC in hypoxic groups are lower than that in the group before hypoxia and decrease progressively with hypoxia time. LDH activities of young CEC in groups after hypoxia and before hypoxia are the same and are apparently lower than that of VEC. Before hypoxia most of CEC are aging, the number of CEC from hypoxic groups is greater than that before hypoxia and increases progressively with hypoxia time and most of CEC from hypoxic groups are young. CONCLUSION: The morphology and number of CEC may reflect the extent to which the vascular is injured. LDH activity of VEC may reflect the transformation from VEC into CEC. LDH activity of young CEC may reflect the extent to which the VEC is injured when falling from vascular wall.