Architecture of the human regulatory network derived from ENCODE data.

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

Tuning out disorder-induced localization in nanophotonic cavity arrays.

Weakly coupled high-Q nanophotonic cavities are building blocks of slow-light waveguides and other nanophotonic devices. Their functionality critically depends on tuning as resonance frequencies should stay within the bandwidth of the device. Unavoidable disorder leads to random frequency shifts which cause localization of the light in single cavities. We present a new method to finely tune individual resonances of light in a system of coupled nanocavities. We use holographic laser-induced heating and address thermal crosstalk between nanocavities using a response matrix approach. As a main result we observe a simultaneous anticrossing of 3 nanophotonic resonances, which were initially split by disorder.

Measurement of the linear thermo-optical coefficient of Ga0.51In0.49P using photonic crystal nanocavities.

Ga0.51In0.49P is a promising candidate for thermally tunable nanophotonic devices due to its low thermal conductivity. In this work we study its thermo-optical response. We obtain the linear thermo-optical coefficient dn/dT=2.0±0.3·10-4 K-1 by investigating the transmission properties of a single mode-gap photonic crystal nanocavity.

Large enchondroma of the thoracic spine: a rare case report and review of the literature.

BACKGROUND: Enchondroma, a subtype of chondroma, originates from the medullary cavity of the bone and produces an expansile growth pattern. Enchondroma located in the spine is rare and a few cases of large thoracic enchondroma have been reported. The authors document a rare case of large enchondroma in the thoracic spine of a 49-year-old woman, and discuss its clinical, radiological and histopathological characteristics. CASE PRESENTATION: The patient presented with rapidly progressive and severe pain on her upper back. Magnetic resonance imaging revealed an expansile lesion at the posterior elements of T3 that was hypointense on T1-weighted images and mixed iso- to hyperintense on T2-weighted images. Administration of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) resulted in heterogeneous enhancement. During surgery, a large tumor of 4.2cm × 4.7cm × 2.1cm was resected along with the lamina and spinous process. Histological examination revealed that the tumor consisted of mature hyaline cartilage with typical chondrocytes, indicating that it was an enchondroma. CONCLUSIONS: Despite its benign-growing nature, enchondroma should be examined closely for signs of enchondromatosis and enchondrosarcoma. Complete surgical resection is the treatment of choice for immediate relief of symptoms and avoidance of recurrence.

Measurement of the profiles of disorder-induced localized resonances in photonic crystal waveguides by local tuning.

Near the band edge of photonic crystal waveguides, localized modes appear due to disorder. We demonstrate a new method to elucidate spatial profile of the localized modes in such systems using precise local tuning. Using deconvolution with the known thermal profile, the spatial profile of a localized mode with quality factor (Q) > 105 is successfully reconstructed with a resolution of 2.5 µm.

Dispersion of coupled mode-gap cavities.

The dispersion of a coupled resonator optical waveguide made of photonic crystal mode-gap cavities is pronouncedly asymmetric. This asymmetry cannot be explained by the standard tight binding model. We show that the fundamental cause of the asymmetric dispersion is the inherent dispersive cavity mode profile; i.e., the mode wave function depends on the driving frequency, not the eigenfrequency. This occurs because the photonic crystal cavity resonances do not form a complete set. We formulate a dispersive mode coupling model that accurately describes the asymmetric dispersion without introducing any new free parameters.

Tcf7 is an important regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line.

A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34- cells, using RNA-Seq. We subsequently identified the target genes bound by TCF7, using ChIP-Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34- state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Hepatoprotective effect of ethanol extract from Berchemia lineate against CCl4-induced acute hepatotoxicity in mice.

CONTEXT: The roots of Berchemia lineate (L.) DC. (Rhamnaceae) have been long used as a remedy for the treatment of some diseases in Guangxi Province, China. OBJECTIVE: The present study investigates the hepatoprotective effect of Berchemia lineate ethanol extract (BELE) on CCl4-induced acute liver damage in mice. MATERIALS AND METHODS: Effect of BELE administrated for 7 consecutive days was evaluated in mice by the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), albulin (ALB), globulin (GLB), and total protein (TP) levels, as well as liver superoxide dismutase (SOD) activity and malondialdehyde (MDA) level. Moreover, histopathological examinations were also taken. RESULTS: Compared with the model group, administration of 400 mg/kg BELE for 7 d in mice significantly decreased the serum ALT (56.25 U/L), AST (297.67 U/L), ALP (188.20 U/L), and TBIL (17.90 mol/L), along with the elevation of TP (64.67 g/L). In addition, BELE (100, 200, and 400 mg/kg, i.g.) treated mice recorded a dose-dependent increment of SOD (291.17, 310.32, and 325.67 U/mg prot) and reduction of MDA (7.27, 6.77, and 5.33 nmol/mg prot) levels. Histopathological examinations also confirmed that BELE can ameliorate CCl4-induced liver injuries, characterized by extensive hepatocellular degeneration/necrosis, inflammatory cell infiltration, congestion, and sinusoidal dilatation. DISCUSSION AND CONCLUSION: The results indicated that BELE possessed remarkable protective effect against acute hepatotoxicity and oxidative injuries induced by CCl4, and that the hepatoprotective effects of BELE may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity.

[Research progress on structure and hydrological processes in the karst critical zone of southwest China]. / è¥¿å—å–€æ–¯ç‰¹å ³é”®å¸¦ç»“æž„åŠå ¶æ°´æ–‡è¿‡ç¨‹ç ”ç©¶è¿›å±•.

The southwestern region of China is the largest exposed karst area in the world and serves as an important ecological security barrier for the upstream of Yangtze River and Pearl River. Different from the critical zone of non-karst areas, the epikarst, formed by an interwoven network of denudation pores, is the core area of karst critical zone. Water is the most active component that participates in internal material cycle and energy flow within the critical zone. We reviewed relevant research conducted in the southwestern region from three aspects: the characte-rization of critical zone structure, the hydrological processes of soil-epikarst system, and their model simulations. We further proposed potential research hotpots. The main approach involved multi-scale and multi-method integrated observations, as well as interdisciplinary collaboration. Precisely characterizing the eco-hydrological processes of the vegetation-soil-epikarst coupling system was a new trend in the future research. This review would provide scientific reference for further studies on hydrological processes in critical zones and regional hydrological water resource management in karst areas.

Screening for diabetic retinopathy with or without a copayment in a randomized controlled trial: influence of the inverse care law.

OBJECTIVE: To examine whether the inverse care law operates in a screening program for diabetic retinopathy (DR) based on fee for service in Hong Kong. DESIGN: Randomized controlled trial. PARTICIPANTS: All those with type 1 or 2 diabetes from 2 clinics were recruited. INTERVENTION: Diabetic retinopathy screening with a small copayment versus free access in a publicly funded family medicine service. MAIN OUTCOME MEASURES: Uptake of screening and severity of DR detected. Association between these outcome variables and independent variables were determined using multivariate logistic regression models and reported as odds ratios (ORs). RESULTS: After randomization, 1387 subjects in the free group and 1379 subjects in the pay group were eligible for screening, and 94.9% (1316/1387) and 92.6% (1277/1379), respectively, agreed to participate in the study. The offer of screening was accepted by 94.8% (1247/1316) in the free group and 91.2% (1164/1277) in the pay group, and the final uptake ratios were 88.5% (1165/1316) and 82.4% (1052/1277), respectively (Pearson chi = 19.74, P<0.001). Being in the pay group was associated with a lower uptake of screening than being in the free group (OR, 0.59; confidence interval [CI], 0.47-0.74) and a lower detection rate of DR (OR, 0.73; CI, 0.60-0.90) after adjustment for potential confounding factors. Subjects with higher socioeconomic status were more likely to attend screening and had a lower prevalence of DR detected. CONCLUSIONS: The inverse care law seems to operate in a preventive intervention when a relatively small copayment is applied. There is a case for making effective preventive services free of charge. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The long-term effect of glutaraldehyde on the bacterial community in anaerobic ammonium oxidation reactor.

A 160-day incubation was performed with two anammox reactors (GA and CK) to investigate the effect of glutaraldehyde. The results indicated that anammox bacteria were very sensitive when glutaraldehyde in GA reactor increased to 40 mg/L, the nitrogen removal efficiency sharply decreased to 11%, only one-quarter of CK. Glutaraldehyde changed spatial distribution of exopolysaccharides, caused anammox bacteria (Brocadia CK_gra75) to disassociate from granules (24.70% of the reads in CK but only 14.09% in GA granules). Metagenome analysis indicated glutaraldehyde led to the denitrifier community succession from strains without nir (nitrite reductase) and nor (nitric oxide reductases) genes to those with them, and the rapid growth of denitrifiers with NodT (an outer membrane factor)-related efflux pumps replacing those with another TolC -related ones. Meanwhile, Brocadia CK_gra75 lacks the NodT proteins. This study provides important insight into community adaptation and potential resistance mechanism in an active anammox community after exposure to disinfectant.

Prospective Comparative Study Investigating Agreement between Tele-Ophthalmology and Face-to-face Consultations in Patients Presenting with Chronic Visual Loss.

INTRODUCTION: This study aims to investigate the diagnostic accuracy of store-and-forward tele-ophthalmology consultations for non-diabetic patients, aged 40 and above, presenting with vision impairment of 3 months or more, in terms of cataracts, glaucoma, and age-related macular degeneration. METHODS: This is a prospective comparative study. Enrolled subjects were independently assessed by both tele-ophthalmology and face-to-face assessment. Agreement level between the two modalities for diagnosis and severity were compared using kappa statistic. Diagnostic accuracy of tele-ophthalmology was determined using the face-to-face consultation serving as the gold standard. Costs were compared by calculating the downstream costs generated by each modality in terms of investigations and treatment. RESULTS: A total of 860 eyes of 430 patients were assessed during the study period. Tele-ophthalmology consultations had significantly high agreement with face-to-face consultations in the diagnosis and grading of all three ocular conditions; cataracts, glaucoma, and AMD. Diagnosis and grading of cataracts and AMD reached [Formula: see text] values of > 0.8, while diagnosis and grading of glaucoma reached [Formula: see text] values between 0.61 and 0.8. In terms of diagnostic accuracy, tele-ophthalmology consultations were highly sensitive and specific for AMD with greater than 99% sensitivity and specificity achieved by tele-ophthalmology. There was high specificity when diagnosing cataracts, but lower sensitivity at 87.8%. Conversely, there was high sensitivity for diagnosing glaucoma, but lower specificity at 76.5%. Downstream costs were similar between groups. CONCLUSIONS: Store-and-forward tele-ophthalmology consultations are accurate and comparable to face-to-face consultations for diagnosis and grading of cataracts, glaucoma, and AMD.

[Clinical Analysis of Bloodstream Infection after Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To analyze the clinical characteristics of bloodstream infection (BSI) in patients treated by hematopoietic stem cell transplantation (HSCT). METHODS: The clinical characteristics, distribution of pathogenic bacteria causing BSI and drug sensitivity of 910 patients treated by HSCT in our department from January 2013 to June 2020 were retrospectively analyzed. RESULTS: Among 910 HSCT patients, 111 patients were diagnosed as BSI within 100 days after transplantation, and 98 patients showed BSI during the period of agranulocytosis. Multivariate analysis showed that the usage of anti-thymocyte globulin (ATG), long duration of agranulocytosis and low infusion volume of mononuclear cell (MNC) were the independent risk factors affecting BSI after HSCT. Among 121 pathogenic bacteria isolated, 76 Gram-negative (G-) bacteria (62.8%), 40 Gram-positive (G+) bacteria (33.0%), and 5 fungi (4.1%) were detected out. The top three pathogens were Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa. The drug-resistance rates of Escherichia coli and Klebsiella pneumoniae to carbapenems was 14.3% and 7.7%, respectively, and Pseudomonas aeruginosa was 66.7%. The susceptibility of G+ bacteria to vancomycin, linezolid and teicoplanin was 97.5%, 100% and 100%, respectively. The crude mortality rate of the patients with BSI at 100 days after HSCT was significantly higher than that of patients without BSI (P<0.001). CONCLUSION: The usage of ATG, long duration of agranulocytosis and low infusion volume of MNC are independent risk factors for BSI after HSCT. The pathogens after HSCT are mainly G- bacteria. Pseudomonas aeruginosa is highly resistant to carbapenems. Key words　　;

[Soil-epikarst structures and their hydrological characteristics on dolomite slopes in karst region of southwest China]. / 西南喀斯特白云岩坡地土壤-表层岩溶带结构及水文特征.

Epikarst is the core area of karst critical zone, with important hydrologic regulation and storage function. However, the effects of karst development degree on hydrologic characteristics of epikasrt is still unclear. We used geophysical exploration and hydrogeological techniques, combined with the dynamic monitoring of moisture and water levels, to quantify the karst development degrees and their hydrologic characteristics on slope lands. We analyzed the responses of soil-epikarst systems to rainfall. Results showed that geophysical exploration technology could be well applied to the detection of surface-subsurface structures in the karst areas. The average thickness of soil and surface karst zone on the slope was less than 0.63 m and 2.60 m, respectively. The slopes of strong-karstification characterized by high apparent resistivity, well-developed joint fractures, and strong permeability (0.73 m·d-1). Such a result indicated that epikarst could regulate precipitation. The responses of soil moisture had a larger rainfall threshold (>20.50 mm·d-1) and the water level was determined by rainfall amount. In contrast, the slope with weak-karstification had low apparent resistivity and weak permeability (0.07 m·d-1). Moisture and water level were sensitive to rainfall. Karst channels were developed locally at 240-300 cm with a permeability coefficient of up to 432 mm·d-1. Obvious preferential flow was observed in extreme rainfall events on this slope, which could induce flood disaster in the adjacent depression. Our results would provide scientific basis for further research on water resources regulation, management, and eco-hydrology in karst areas of southwest China.

Investigation on the Performance of Partial Penetration Welds in Multicell Concrete Filled Steel Tubes.

This paper presents an experimental and analytical investigation on the performance of partial penetration welds used to adjoin steel plates in irregular shaped multicell concrete filled steel tubes. The experimental program of this study is designed based on an actual implementation of such members as mega columns in a super high rise building in China. A total of six specimens are designed with different plate arrangements for the purpose of testing the performance of the partial penetration welds at different locations of the specimen. The designed specimens are tested under different load procedures and directions; this is achieved by placing them in vertical and slantwise manners between two loading plates which impose monotonic and cyclic actions. The failure conditions of each of the tested specimens are presented and discussed in detail and are based on the conclusions drawn from the experimental observations; the partial penetration weld at the corner of the tested specimens is found to be the most vulnerable. To facilitate large scale analysis, a finite element model constructed by the finite element analysis program ABAQUS is verified against experimental results. The evaluation of the stress at the partial penetration welded corner is carried out following an empirical procedure, which is adopted due to the complexity of the problem domain. The adopted procedure consists of two steps: the first one is to initially evaluate the stress based on an existing method in the literature, and the second one is to fit the results of the initial evaluation with the finite element model results based on parametric and regression analysis. After performing regression analysis, a formula to predict the weld stress is concluded, and the results of the proposed equation are found to be satisfactory when compared with the finite element model results.

Screening for diabetic retinopathy with different levels of financial incentive in a randomized controlled trial.

AIMS/INTRODUCTION: To examine the impact of different levels of financial incentive in terms of fee subsidization on diabetic retinopathy screening in the private primary care setting in Hong Kong. MATERIALS AND METHODS: All general practitioners working in the private sector and registered in two electronic public databases were invited to participate. Consecutive patients with diabetes mellitus were then recruited by the participating practitioners. The recruited participants were randomly allocated to one of three screening groups with different fee levels (HK$0, HK$150 [US$19], HK$300 [US$39]) in a randomized controlled trial. Screening uptake and severity of diabetic retinopathy detected were compared. RESULTS: Out of 1,688 eligible practitioners, 105 participated and invited 402 patients, with 239 initially agreeing to participate (59.5%). After randomization, 78, 75 and 76 participants in the HK$0, HK$150 and HK$300 fee groups, respectively, reconfirmed their participation and were offered screening at the relevant fee. The uptake of screening was 79.5% (62/78), 81.3% (61/75) and 63.2% (48/76), in the HK$0, HK$150 and HK$300 groups, respectively (P < 0.018). Being in the HK$150 fee group was associated with higher uptake of screening than being in the HK$300 fee group (odds ratio 2.31, P = 0.039). No significant difference was found in the prevalence of any diabetic retinopathy (33.9%, 27.9% and 37.5%, P = 0.378) or sight-threatening diabetic retinopathy (4.8%, 8.2% and 16.7%; P = 0.092) among the groups. CONCLUSION: A screening fee of HK$150, representing approximately a half subsidy, appears to be as effective in maximizing uptake as a full subsidy (HK$0) and without deterring those at high risk of diabetic retinopathy from screening.

Use of the BCR sequential extraction procedure for the study of metal availability to plants.

To investigate the mobility and availability of metals from soil to plant, concentrations of zinc (Zn), copper (Cu), lead (Pb), cadmium (Cd), mercury (Hg) and arsenic (As) in topsoils and plants (lettuce, scallion, celery, tomato, carambola, wampee and longan) collected from the area around a petrochemical complex in Guangzhou, China, were analyzed. The modified European Community Bureau of Reference (BCR) three-step sequential extraction procedure was applied to determine the concentration of metal fractions in soils. The results showed that the distribution of Zn, Cu, Pb and Cd in four fractions varied greatly among the soil samples, and 18.8% of vegetable and fruit samples for Cd and 5.8% for Pb exceeded the maximum permissible levels in food of China. Soil-to-plant transfer coefficients were in the order of Cd>Zn>Cu>Hg>As>Pb, suggesting Cd being the most mobile and available to plants among the metals studied. Principal component analysis indicated that metal fractions and soil physicochemical properties (pH, organic matter, cation exchange capacity, clay content and electrical conductivity) affected metal uptake by plants. Furthermore, atmospheric deposition may be another important factor for the accumulation of metals in plants.

[Dual regulation effect of somatostatin on immunity in patients with severe sepsis caused by abdominal diseases].

OBJECTIVE: to investigate the effect of somatostatin on inflammatory immune disorders and prognosis in patients with severe sepsis caused by abdominal diseases. METHODS: fifty-three patients with severe abdominal sepsis (age > 18 years, APACHE-II score > 15) from June 2005 to June 2009 were randomly divided into Somatostatin group (n = 23) and SSC Group (n = 30). Fifteen healthy volunteers of the same age range were chosen as Control group. The SSC group was treated with classical SSC therapy, and the Somatostatin Group was treated with the same regime plus 14-peptide somatostatin continuous infusion at the dose of 6 mg/24 h for 7 days. The serum levels of interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) were determined by using ELISA. CD(4)(+), CD(8)(+) T cell subsets were determined by fluorescence activated cell sorter(FACS) and CD(4)(+)/CD(8)(+) was calculated. APACHE-II score was observed on admission (d1) and day 3, 7 and 14 after treatment. Morality rates in 28 days in two groups were recorded. RESULTS: compared with Control group, IL-10 and TNF-α levels were significantly elevated in patients with severe abdominal sepsis (P < 0.05), while CD(4)(+), CD(8)(+) T cell and CD(4)(+)/CD(8)(+) decreased significantly (P < 0.05). Compared with the Somatostatin group CD(4)(+), CD(8)(+) T cell and CD(4)(+)/CD(8)(+) on d7 and d14 in SSC Group were significantly increased (P < 0.05), while IL-10 and TNF-α decreased significantly(P < 0.05). APACHE-II scores on d3, d7, d14 of Somatostatin group were significantly lower than those of SSC group, and 28 d mortality rate also declined. CONCLUSIONS: in patients with severe abdominal sepsis, systemic inflammatory response and immune suppression exist simultaneously. Somatostatin has a dual immunomodulatory activity in these patients.

Diagnostic value of radionuclide in bone metastasis after breast cancer surgery: A protocol of systematic review.

BACKGROUND: The objective of this study is to evaluate the accuracy of radionuclide in diagnosis of bone metastasis (BM) after breast cancer surgery (BCS). METHODS: The electronic databases (Cochrane Library, MEDLINE, EMBASE, Web of Science, CBM, and CNKI) will be systematically and comprehensively searched until June 1, 2020 for eligible studies that reported the diagnosis of radionuclide in BM after BCS. In addition, we will also identify grey literatures, such as conference abstracts, and reference lists of included studies. All process of study identification, data extraction, and study methodological quality evaluation will be performed by 2 independent authors. All divergences will be settled by a third author through discussion. All data analysis will be carried out by RevMan 5.3 software (London, UK). RESULTS: This study will scrutinize the most recent evidence of radionuclide in detection of BM after BCS. CONCLUSION: This study may provide evidence of accuracy of radionuclide in diagnosis of BM following BCS. STUDY REGISTRATION NUMBER: PROSPERO CRD42020187646.