Physico-chemical characterization and possible uses of sludge processed from an urban sewage treatment plant.

Nowadays, the challenge is to transform dehydrated sewage sludge resulting from wastewater treatment plants from waste into resource. Following this objective, the sludge was further dried and submitted to X-ray diffraction (XRD) and FTIR analysis. The sludge was first dried in ventilated and unventilated spaces at 50 ∘C and 100 ∘C, for 60 and 100 minutes (min) in each case. The final mass and evaporation degree of the sludge depends on the initial mass, ventilation type, drying time, and temperature. The ventilated drying space is preferred for temperature control, homogeneity, and higher evaporation degree. The influence of the drying process on the structure and behavior of the sewage sludge was emphasized through X-ray diffraction (XRD) and FTIR analysis. The XRD shows good structural properties of the sludge samples given by the reduction of the particle size through evaporation. According to FTIR, evaporation influences the depolymerization of the silicate network. The hydroxyl units and metallic ion modifiers can improve the sludge structure, but its intensity decreases through evaporation. With high content of solid substance, and good relation between the composition of the sludge and its structure and behavior, the dried sewage sludge can be used in: (i) agriculture, (ii) construction, (iii) the energy sector.

Frailty in acute coronary syndromes. A systematic review and narrative synthesis of frailty assessment tools and interventions from randomised controlled trials.

AIM: We aimed to review all randomised controlled trial (RCT) data to explore optimal identification and treatment strategies of frail patients with Acute Coronary Syndromes (ACS). METHODS: The protocol was preregistered (PROSPERO - CRD42021250235). We performed a systematic review including RCT's that 1; used at least one frailty assessment tool to assess frailty and its impact on outcomes in patients diagnosed with ACS and 2; used at least one intervention where change in frailty was measured in patients diagnosed with ACS. The Cochrane Central Register of Controlled Trials, MEDLINE and EMBASE were searched on the 1st April 2021 and updated on 4th July 2023. Owing to low search output results are presented as a narrative synthesis of available evidence. RESULTS: A single RCT used a frailty assessment tool. A single RCT specifically targeted frailty with their intervention. This precluded further quantitative analysis. There was indication of selection bias against frail participants, and a signal of value for physical activity measurement in frail ACS patients. There was a high level of uncertainty and low level of robustness of this evidence. CONCLUSIONS: Data from RCT's alone is inadequate in answering the reviews question. Future RCT's need to address ways to incorporate frail participants, whilst mitigating selection biases. Physical performance aspects of the frailty syndrome appear to be high yield modifiable targets that improve outcomes. Intervention trials should consider using change in frailty status as an outcome measure. Any trials that include frail participants should present data specifically attributable to this group.

Effects of interventions targeting the systemic inflammatory response to cardiac surgery on clinical outcomes in adults.

BACKGROUND: Organ injury is a common and severe complication of cardiac surgery that contributes to the majority of deaths. There are no effective treatment or prevention strategies. It has been suggested that innate immune system activation may have a causal role in organ injury. A wide range of organ protection interventions targeting the innate immune response have been evaluated in randomised controlled trials (RCTs) in adult cardiac surgery patients, with inconsistent results in terms of effectiveness. OBJECTIVES: The aim of the review was to summarise the results of RCTs of organ protection interventions targeting the innate immune response in adult cardiac surgery. The review considered whether the interventions had a treatment effect on inflammation, important clinical outcomes, or both. SEARCH METHODS: CENTRAL, MEDLINE, Embase, conference proceedings and two trial registers were searched on October 2022 together with reference checking to identify additional studies. SELECTION CRITERIA: RCTs comparing organ protection interventions targeting the innate immune response versus placebo or no treatment in adult patients undergoing cardiac surgery where the treatment effect on innate immune activation and on clinical outcomes of interest were reported. DATA COLLECTION AND ANALYSIS: Searches, study selection, quality assessment, and data extractions were performed independently by pairs of authors. The primary inflammation outcomes were peak IL-6 and IL-8 concentrations in blood post-surgery. The primary clinical outcome was in-hospital or 30-day mortality. Treatment effects were expressed as risk ratios (RR) and standardised mean difference (SMD) with 95% confidence intervals (CI). Meta-analyses were performed using random effects models, and heterogeneity was assessed using I2. MAIN RESULTS: A total of 40,255 participants from 328 RCTs were included in the synthesis. The effects of treatments on IL-6 (SMD -0.77, 95% CI -0.97 to -0.58, I2 = 92%) and IL-8 (SMD -0.92, 95% CI -1.20 to -0.65, I2 = 91%) were unclear due to heterogeneity. Heterogeneity for inflammation outcomes persisted across multiple sensitivity and moderator analyses. The pooled treatment effect for in-hospital or 30-day mortality was RR 0.78, 95% CI 0.68 to 0.91, I2 = 0%, suggesting a significant clinical benefit. There was little or no treatment effect on mortality when analyses were restricted to studies at low risk of bias. Post hoc analyses failed to demonstrate consistent treatment effects on inflammation and clinical outcomes. Levels of certainty for pooled treatment effects on the primary outcomes were very low. AUTHORS' CONCLUSIONS: A systematic review of RCTs of organ protection interventions targeting innate immune system activation did not resolve uncertainty as to the effectiveness of these treatments, or the role of innate immunity in organ injury following cardiac surgery.

The impact of the COVID-19 outbreak on emergency general surgery in the first German "hotspot region" Aachen-Heinsberg-A multicentre retrospective cohort study.

This study aimed to observe the impact of the COVID-19 outbreak on acute general surgery in the first German "hotspot" regions of Heinsberg and Aachen, during the first months of the pandemic. The incidence and severity of acute appendicitis, acute cholecystitis and mechanical bowel obstruction, were compared between March and May 2020 and a control period (same months of the previous three years). Pre-, intra- and postoperative data was compared between three regional hospitals of Heinsberg and the closest maximum care, university hospital. A total of 592 operated patients were included, 141 belonging to the pandemic cohort and 451 to the historic cohort. The pandemic group showed higher rates of clinical peritonitis (38% vs. 27%, p = 0.015), higher rates of mean white blood cell count (13.2±4.4 /nl vs. 12.3±4.7 /nl, p = 0.044) and mean C-reactive protein (60.3±81.1 mg/l vs. 44.4±72.6 mg/l, p = 0.015) preoperatively. Specifically in patients with acute appendicitis, there were less patients with catarrhal appendicitis (23% vs. 35%, p = 0.021) and a tendency towards more advanced histological findings in the pandemic cohort. In the university hospital, a 42% reduction in acute operated cases was observed at the onset of the pandemic (n = 30 in 2020 vs. n = 52 in 2019), whereas in the peripheral hospitals of Heinsberg there was only a 10% reduction (n = 111 in 2020 vs. n = 123 in 2019). The onset of the COVID-19 pandemic in our region was accompanied by advanced preoperative and intraoperative findings in patients undergoing emergency general surgery. A greater reduction in acute operated surgical cases was observed at the university hospital, in contrast to the smaller hospitals of Heinsberg, suggesting a possible shift of emergency patients, requiring immediate operation, from maximum care hospital to the periphery.

Surgical or percutaneous coronary revascularization for heart failure: an in silico model using routinely collected health data to emulate a clinical trial.

AIMS: The choice of revascularization with coronary artery bypass grafting (CABG) vs. percutaneous coronary intervention (PCI) in people with ischaemic left ventricular dysfunction is not guided by high-quality evidence. METHODS AND RESULTS: A trial of CABG vs. PCI in people with heart failure (HF) was modelled in silico using routinely collected healthcare data. The in silico trial cohort was selected by matching the target trial cohort, identified from Hospital Episode Statistics in England, with individual patient data from the Surgical Treatment for Ischemic Heart Failure (STICH) trial. Allocation to CABG vs. complex PCI demonstrated random variation across administrative regions in England and was a valid statistical instrument. The primary outcome was 5-year all-cause mortality or cardiovascular hospitalization. Instrumental variable analysis (IVA) was used for the primary analysis. Results were expressed as average treatment effects (ATEs) with 95 confidence intervals (CIs). The target population included 13 519 HF patients undergoing CABG or complex PCI between April 2009 and March 2015. After matching, the emulated trial cohort included 2046 patients. The unadjusted primary outcome rate was 51.1 in the CABG group and 70.0 in the PCI group. IVA of the emulated cohort showed that CABG was associated with a lower risk of the primary outcome (ATE 16.2, 95 CI 20.6 to 11.8), with comparable estimates in the unmatched target population (ATE 15.5, 95 CI 17.5 to 13.5). CONCLUSION: In people with HF, in silico modelling suggests that CABG is associated with fewer deaths or cardiovascular hospitalizations at 5 years vs. complex PCI. A pragmatic clinical trial is needed to test this hypothesis and this trial would be feasible.

Biological Activated Sludge from Wastewater Treatment Plant before and during the COVID-19 Pandemic.

The COVID-19 pandemic and the related measures brought a change in daily life that affected the characteristics of the municipal wastewater and further, of the biological activated sludge. The activated sludge process is the most widely used biological wastewater treatment process in developed areas. In this paper, we aim to show the situation of specific investigations concerning the variation of the physicochemical parameters and biological composition of the activated sludge from one conventional wastewater treatment plant from a metropolitan area. The investigations were carried out for three years: 2019, 2020 and 2021. The results showed the most representative taxa of microorganisms: Microtrix, Aspidisca cicada, Vorticella convallaria, Ciliata free of the unknown and Epistylis and Rotifers. Even if other microorganisms were found in the sludge flocs, their small presence did not influence in any way the quality of the activated sludge and of the wastewater treatment process. That is why we conclude that protozoa (especially Flagellates and Ciliates) and rotifers were the most important. Together with the values and variation of the physicochemical parameters, they indicated a good, healthy, and stable activated sludge, along with an efficient purifying treatment process, no matter the loading conditions.

Reporting conflicts of interest in randomised trials of patient blood management interventions in patients requiring major surgery: a systematic review and meta-analysis.

OBJECTIVE: This study aimed to systematically review the effects of declared and undeclared conflicts of interest on randomised controlled trials (RCTs) of patient blood management (PBM) interventions. DESIGN: We performed a secondary analysis of a recently published meta-analysis of RCTs evaluating five common PBM interventions in patients undergoing major surgery. DATA SOURCES: The databases searched by the original systematic reviews were searched using subject headings and Medical Subject Headings terms according to search strategies from the final search time-points until 1 June 2019. ELIGIBILITY CRITERIA: RCTs on PBM irrespective of blinding, language, date of publication and sample size were included. Abstracts and unpublished trials were excluded. Conflicts of interest were defined as sponsorship, funding or authorship by industry, professional PBM advocacy groups or blood services. DATA EXTRACTION AND SYNTHESIS: Three independent reviewers extracted the data and assessed the risk of bias. Pooled treatment effect estimates were reported as risk ratios (RRs) or standardised mean difference with 95% CIs. Heterogeneity was quantified using the I2 statistic. RESULTS: Three hundred and eighty-nine RCTs totalling 53 635 participants were included. Thirty-two trials (8%) were considered free from important sources of bias. There was reporting bias favouring PBM interventions on transfusion across all analyses. In trials with no declared author conflicts of interest, the treatment effect on mortality was RR 1.12 (0.86 to 1.45). In trials where author conflicts of interest were declared, the treatment effect on mortality was RR 0.84 (0.69 to 1.03), with significant reporting bias favouring PBM interventions. Trials with declared conflicts linked to professional PBM advocacy groups (five studies, n=977 patients) reported statistically significant reductions in mortality RR 0.40 (0.17 to 0.92), unlike other groups. CONCLUSIONS: Low certainty of the evidence that guides PBM implementation is confounded by evidence of reporting bias, and the effects of declared and undeclared conflicts of interest, favouring PBM on important trial outcomes.

Urinary extracellular vesicles and micro-RNA as markers of acute kidney injury after cardiac surgery.

We hypothesised that measuring changes in urinary levels of EV and miR will predict the onset of acute kidney injury in cardiac surgery patients. The study was performed in the cohort of the REVAKI-2 trial. Urine samples were collected before and 24 h after the procedure from 94 cardiac surgery patients. Urinary particle concentrations and size distribution were assessed using NanoSight. EV derivation and levels were measured using flow cytometry. Samples from 10 selected patients were sequenced, and verification was performed with advanced TaqMan assays in samples from all patients. Urinary particle concentrations significantly increased in patients with AKI after surgery, with the percentage of EV positive for CD105 and ß1-integrin also increasing. Pre-surgery podocalyxin-positive EV were significantly lower in patients with AKI. Their levels correlated with the severity of the injury. Pre-operative miR-125a-5p was expressed at lower levels in urine from patients with AKI when adjusted for urinary creatinine. Levels of miR-10a-5p were lower after surgery in AKI patients and its levels correlated with the severity of the injury. Pre-operative levels of podocalyxin EVs, urinary particle concentrations and miR-125a-5p had moderate AKI predictive value and, in a logistic model together with ICU lactate levels, offered good (AUC = 82%) AKI prediction.

Characteristics of Volcanic Tuff from Macicasu (Romania) and Its Capacity to Remove Ammonia from Contaminated Air.

In the present work, the capability of the volcanic tuff from Macicasu (Romania) to remove ammonia (NH3) from air with different contamination levels during 24 h of adsorption experiments was investigated. The natural zeolitic volcanic tuff was characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), the Brunauer-Emmett-Teller (BET) method, inductively coupled plasma optical emission spectrometry (ICP-OES), and thermogravimetric analysis (TGA). The adsorption capacities varied between 0.022 mg NH3 g-1 zeolite and 0.282 mg NH3 g-1 zeolite, depending on the NH3 concentrations in the air and at the contact time. The nonlinear forms of the Langmuir and Freundlich isotherm models were used to fit the experimental data. Additionally, the adsorption of NH3 was studied using nonlinear pseudo-first-order (PFO), pseudo-second-order (PSO), and Elovich kinetic model. Based on the total volume of pores of used volcanic tuff, the NH3 was removed from the air both due to the physical adsorption of NH3 gas and the ion exchange of NH4+ (resulted from a reaction between NH3 and H2O adsorbed by the zeolite). Depending on the initial NH3 concentration and the amount of volcanic tuff, the NH3 concentrations can be reduced below the threshold of this contaminant in the air. The adsorption capacity of NH3 per unit of zeolite (1 g) varied in the range of 0.022-0.282 mg NH3 g-1 depending on the NH3 concentration in the air.

Gene and metabolite expression dependence on body mass index in human myocardium.

We hypothesized that body mass index (BMI) dependent changes in myocardial gene expression and energy-related metabolites underlie the biphasic association between BMI and mortality (the obesity paradox) in cardiac surgery. We performed transcriptome profiling and measured a panel of 144 metabolites in 53 and 55, respectively, myocardial biopsies from a cohort of sixty-six adult patients undergoing coronary artery bypass grafting (registration: NCT02908009). The initial analysis identified 239 transcripts with biphasic BMI dependence. 120 displayed u-shape and 119 n-shape expression patterns. The identified local minima or maxima peaked at BMI 28-29. Based on these results and to best fit the WHO classification, we grouped the patients into three groups: BMI < 25, 25 ≤ BMI ≤ 32, and BMI > 32. The analysis indicated that protein translation-related pathways were downregulated in 25 ≤ BMI ≤ 32 compared with BMI < 25 patients. Muscle contraction transcripts were upregulated in 25 ≤ BMI ≤ 32 patients, and cholesterol synthesis and innate immunity transcripts were upregulated in the BMI > 32 group. Transcripts involved in translation, muscle contraction and lipid metabolism also formed distinct correlation networks with biphasic dependence on BMI. Metabolite analysis identified acylcarnitines and ribose-5-phosphate increasing in the BMI > 32 group and α-ketoglutarate increasing in the BMI < 25 group. Molecular differences in the myocardium mirror the biphasic relationship between BMI and mortality.

Activation of the innate immune response and organ injury after cardiac surgery: a systematic review and meta-analysis of randomised trials and analysis of individual patient data from randomised and non-randomised studies.

BACKGROUND: It is unclear whether the innate immune response represents a therapeutic target for organ protection strategies in cardiac surgery. METHODS: A systematic review of trials of interventions targeting the inflammatory response to cardiac surgery reporting treatment effects on both innate immune system cytokines and organ injury was performed. The protocol was registered at the International Prospective Register of Systematic Reviews: CRD42020187239. Searches of the Cochrane Central Register of Controlled Trials, MEDLINE, and Embase were performed. Random-effects meta-analyses were used for the primary analysis. A separate analysis of individual patient data from six studies (n=785) explored sources of heterogeneity for treatment effects on cytokine levels. RESULTS: Searches to May 2020 identified 251 trials evaluating 24 interventions with 20 582 participants for inclusion. Most trials had important limitations. Methodological limitations of the included trials and heterogeneity of the treatment effects on cytokine levels between trials limited interpretation. The primary analysis demonstrated inconsistency in the direction of the treatment effects on innate immunity and organ failure or death between interventions. Analyses restricted to important subgroups or trials with fewer limitations showed similar results. Meta-regression, pooling available data from all trials, demonstrated no association between the direction of the treatment effects on inflammatory cytokines and organ injury or death. The analysis of individual patient data demonstrated heterogeneity in the association between the cytokine response and organ injury after cardiac surgery for people >75 yr old and those with some chronic diseases. CONCLUSIONS: The certainty of the evidence for a causal relationship between innate immune system activation and organ injury after cardiac surgery is low.

Evaluation of the Impact of Different Natural Zeolite Treatments on the Capacity of Eliminating/Reducing Odors and Toxic Compounds.

Unlike odorants that mask odors, natural zeolite acts as a molecular sieve that captures and eliminates odors. Different treatment methods can be applied to influence the properties of the natural zeolites. To enhance the odor adsorption capacities of the natural zeolite two types of treatment methods were applied: chemical (acid, basic) and thermal. The initial natural zeolites and the activated one were characterized using X-ray diffraction (XRD) and scanning electron microscope (SEM-EDX). Two experiments were performed to establish the odor adsorption capacity of the activated natural zeolites. The best zeolite for the adsorption of humidity, ammonia and hydrogen sulfide was the 1-3 mm zeolite activated through thermal treatment. For the adsorption of PAHs, the best zeolite was the one activated through basic treatment, with an adsorption capacity of 89.6 ng/g.

Type A aortic syndromes in COVID-19 positive patients: Case series from a UK multicentre study.

OBJECTIVE: The coronavirus disease (COVID-19) increases the respiratory complications and carries a higher mortality in the immediate postoperative period. The aim of this study was to analyze the outcomes of patients with type A acute aortic syndromes (AAS) diagnosed with COVID-19 in the perioperative period. METHODS: Retrospective analysis of prospectively collected data between March and August 2020 from 20 participating cardiac surgery centers in the United Kingdom. RESULTS: Among 122 patients who underwent emergency surgical repair for type A AAS at the participating centers, 3 patients (2.5%) tested positive for COVID-19 in the preoperative screeing, and 4 cases turned to be positive in the postoperative period having been operated on an unknown COVID-19 status. Another patient was diagnosed of COVID-19 disease based on radiological features. These eight patients had increased postoperative complications, including respiratory failure, longer ventilation times, and Intensive Therapy Unit (ITU) stay and increased mortality when compared with COVID-19 negative patients. CONCLUSION: COVID-19 disease in the perioperative period of type A AAS poses a challenge. Despite the increasing morbidity and mortality associated with the virus, the prognosis of the aortic disease is poorer and emergency surgery should not be contraindicated based on the COVID-19 diagnosis alone.

Pharmacological interventions for the prevention of renal injury in surgical patients: a systematic literature review and meta-analysis.

BACKGROUND: The aim of this systematic review was to summarise the results of randomised controlled trials (RCTs) that have evaluated pharmacological interventions for renoprotection in people undergoing surgery. METHODS: Searches were conducted to update a previous review using the Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE to August 23, 2019. RCTs evaluating the use of pharmacological interventions for renal protection in the perioperative period were included. The co-primary outcome measures were 30-day mortality and acute kidney injury (AKI). Pooled effect estimates were expressed as risk ratios (RRs) (95% confidence intervals). RESULTS: We included 228 trials enrolling 56 047 patients. Twenty-three trials were considered to be at low risk of bias across all domains. Atrial natriuretic peptides (14 trials; n=2207) reduced 30-day mortality (RR: 0.63 [0.41, 0.97]) and AKI events (RR: 0.43 [0.33, 0.56]) without heterogeneity. These effects were consistent across cardiac surgery and vascular surgery subgroups, and in sensitivity analyses restricted to studies at low risk of bias. Inodilators (13 trials; n=2941) reduced mortality (RR: 0.71 [0.53, 0.94]) and AKI events (RR: 0.65 [0.50, 0.85]) in the primary analysis and in cardiac surgery cohorts. Vasopressors (4 trials; n=1047) reduced AKI (RR: 0.56 [0.36, 0.86]). Nitric oxide donors, alpha-2-agonists, and calcium channel blockers reduced AKI in primary analyses, but not after exclusion of studies at risk of bias. Overall, assessment of the certainty of the effect estimates was low. CONCLUSIONS: There are multiple effective pharmacological renoprotective interventions for people undergoing surgery.

Patient blood management interventions do not lead to important clinical benefits or cost-effectiveness for major surgery: a network meta-analysis.

BACKGROUND: Patient blood management (PBM) interventions aim to improve clinical outcomes by reducing bleeding and transfusion. We assessed whether existing evidence supports the routine use of combinations of these interventions during and after major surgery. METHODS: Five systematic reviews and a National Institute of Health and Care Excellence health economic review of trials of common PBM interventions enrolling participants of any age undergoing surgery were updated. The last search was on June 1, 2019. Studies in trauma, burns, gastrointestinal haemorrhage, gynaecology, dentistry, or critical care were excluded. The co-primary outcomes were: risk of receiving red cell transfusion and 30-day or hospital all-cause mortality. Treatment effects were estimated using random-effects models and risk ratios (RR) with 95% confidence intervals (CIs). Heterogeneity assessments used I2. Network meta-analyses used a frequentist approach. The protocol was registered prospectively (PROSPERO CRD42018085730). RESULTS: Searches identified 393 eligible randomised controlled trials enrolling 54 917 participants. PBM interventions resulted in a reduction in exposure to red cell transfusion (RR=0.60; 95% CI 0.57, 0.63; I2=77%), but had no statistically significant treatment effect on 30-day or hospital mortality (RR=0.93; 95% CI 0.81, 1.07; I2=0%). Treatment effects were consistent across multiple secondary outcomes, sub-groups and sensitivity analyses that considered clinical setting, type of intervention, and trial quality. Network meta-analysis did not demonstrate additive benefits from the use of multiple interventions. No trial demonstrated that PBM was cost-effective. CONCLUSIONS: In randomised trials, PBM interventions do not have important clinical benefits beyond reducing bleeding and transfusion in people undergoing major surgery.

A peritoneal defect covered by intraperitoneal mesh prosthesis effects an increased and distinctive foreign body reaction in a minipig model.

BACKGROUND: The incidence of incisional hernia is with up to 30% one of the frequent long-term complication after laparotomy. After establishing minimal invasive operations, the laparoscopic intraperitoneal onlay mesh technique (lap. IPOM) was first described in 1993. Little is known about the foreign body reaction of IPOM-meshes, which covered a defect of the parietal peritoneum. This is becoming more important, since IPOM procedure with peritoneal-sac resection and hernia port closing (IPOM plus) is more frequently used. METHODS: In 18 female minipigs, two out of three Polyvinylidene-fluoride (PVDF) -meshes (I: standard IPOM; II: IPOM with modified structure [bigger pores]; III: IPOM with the same structure as IPOM II + degradable hydrogel-coating) were placed in a laparoscopic IPOM procedure. Before mesh placement, a 2x2cm peritoneal defect was created. After 30 days, animals were euthanized, adhesions were evaluated by re-laparoscopy and mesh samples were explanted for histological and immunohistochemichal investigations. RESULTS: All animals recovered after implantation and had no complications during the follow-up period. Analysing foreign body reaction, the IPOM II mesh had a significant smaller inner granuloma, compared to the other meshes (IPOM II: 8.4 µm ± 1.3 vs. IPOM I 9.1 µm ± 1.3, p < 0.001). The degradable hydrogel coating does not prevent adhesions measured by Diamond score (p = 0.46). A peritoneal defect covered by a standard or modified IPOM mesh was a significant factor for increasing foreign body granuloma, the amount of CD3+ lymphocytes, CD68+ macrophages and decrease of pore size. CONCLUSION: A peritoneal defect covered by IPOM prostheses leads to an increased foreign body reaction compared to intact peritoneum. Whenever feasible, a peritoneal defect should be closed accurately before placing an IPOM-mesh to avoid an excessive foreign body reaction and therefore inferior biomaterial properties of the prosthesis.