Cytochrome b5 reductase 3 overexpression and dietary nicotinamide riboside supplementation promote distinctive mitochondrial alterations in distal convoluted tubules of mouse kidneys during aging.

The kidney undergoes structural and physiological changes with age, predominantly studied in glomeruli and proximal tubules. However, limited knowledge is available about the impact of aging and anti-aging interventions on distal tubules. In this study, we investigated the effects of cytochrome b5 reductase 3 (CYB5R3) overexpression and/or dietary nicotinamide riboside (NR) supplementation on distal tubule mitochondria. Initially, transcriptomic data were analyzed to evaluate key genes related with distal tubules, CYB5R3, and NAD+ metabolism, showing significant differences between males and females in adult and old mice. Subsequently, our emphasis focused on assessing how these interventions, that have demonstrated the anti-aging potential, influenced structural parameters of distal tubule mitochondria, such as morphology and mass, as well as abundance, distance, and length of mitochondria-endoplasmic reticulum contact sites, employing an electron microscopy approach. Our findings indicate that both interventions have differential effects depending on the age and sex of the mice. Aging resulted in an increase in mitochondrial size and a decrease in mitochondrial abundance in males, while a reduction in abundance, size, and mitochondrial mass was observed in old females when compared with their adult counterparts. Combining both the interventions, CYB5R3 overexpression and dietary NR supplementation mitigated age-related changes; however, these effects were mainly accounted by NR in males and by transgenesis in females. In conclusion, the influence of CYB5R3 overexpression and dietary NR supplementation on distal tubule mitochondria depends on sex, genotype, and diet. This underscores the importance of incorporating these variables in subsequent studies to comprehensively address the multifaceted aspects of aging.

High throughput screening aids clinical decision-making in refractory acute myeloid leukaemia.

BACKGROUND: Despite advances in therapeutics for adverse-risk acute myeloid leukaemia (AML), overall survival remains poor, especially in refractory disease. Comprehensive tumour profiling and pre-clinical drug testing can identify effective personalised therapies. CASE: We describe a case of ETV6-MECOM fusion-positive refractory AML, where molecular analysis and in vitro high throughput drug screening identified a tolerable, novel targeted therapy and provided rationale for avoiding what could have been a toxic treatment regimen. Ruxolitinib combined with hydroxyurea led to disease control and enhanced quality-of-life in a patient unsuitable for intensified chemotherapy or allogeneic stem cell transplantation. CONCLUSION: This case report demonstrates the feasibility and role of combination pre-clinical high throughput screening to aid decision making in high-risk leukaemia. It also demonstrates the role a JAK1/2 inhibitor can have in the palliative setting in select patients with AML.

Simultaneous detection of IgG, IgM, IgA complexes and C3d attached to erythrocytes by flow cytometry.

INTRODUCTION: Immune complexes attached to erythrocyte membrane are involved in autoimmune hemolytic anemia (AIHA) pathogenesis. Currently, direct antiglobulin test (DAT) is used for AIHA diagnosis; however, its performance can be variable. The aim of this study was to design a flow cytometry protocol for simultaneous detection of IgG, IgM, IgA immune complexes and C3d attached to erythrocytes in AIHA patients . METHODS: A procedure was standardized for assessing independent or simultaneous IgG, IgM, IgA immune complexes and C3d, which were detected using secondary antibodies. The protocol developed was applied to blood samples of patients with AIHA, donors at risk of developing the disease, and healthy controls. RESULTS: Twenty-four blood samples were assessed: nine patients with AIHA, five donors at risk of developing the disease, and 10 healthy controls. In the AIHA group, all were positive for C3d, seven for IgG, four for IgA, and one for IgM. Two AIHA patients that were negative for DAT-IgG and C3d were positive for C3d by flow cytometry. CONCLUSION: Flow cytometry is a consistent method for identifying the presence of IgG, IgM, IgA immune complexes and C3d attached to erythrocytes and can be helpful for understanding the mechanisms involved in AIHA pathogenesis.

Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes.

AIMS/HYPOTHESIS: We previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marrow-derived cells are also involved in this HGF-induced reparative process. METHODS: We have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals. RESULTS: We find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman's capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation. CONCLUSIONS/INTERPRETATION: Altogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

Frequency of specific CD8+ T cells for a promiscuous epitope derived from Trypanosoma cruzi KMP-11 protein in chagasic patients.

The K1 peptide is a CD8(+)T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein. We have previously shown that this peptide induces IFN-gamma secretion by CD8(+)T cells. The aim of this study was to characterize the frequency of K1-specific CD8(+)T cells in chagasic patients. Nineteen HLA-A2(+)individuals were selected from 50 T. cruzi infected patients using flow cytometry and SSP-PCR assays. Twelve HLA-A*0201(+)noninfected donors were included as controls. Peripheral blood mononuclear cells were stained with HLA-A2-K1 tetramer, showing that 15 of 19 infected patients have K1-specific CD8(+)T cells (0.09-0.34% frequency) without differences in disease stages or severity. Of note, five of these responders were A*0205, A*0222, A*0226, A*0259 and A*0287 after molecular typing. Thus, a phenotypic and functional comparison of K1-specific CD8(+)T cells from non-HLA-A*0201 and HLA-A*0201(+)infected patients was performed. The results showed that both non-HLA-A*0201 and HLA-A*0201(+)individuals have a predominant effector memory CD8(+)T cell phenotype (CCR7-, CD62L-). Moreover, CD8(+)T cells from non-HLA-A*0201 and HLA-A*0201(+)individuals expressed IL-2, IFN-gamma and perforin without any differences. These findings support that K1 peptide is a promiscuous epitope presented by HLA-A2 supertype molecules and is highly recognized by chagasic patients.

New immunosuppresor strategies in the treatment of murine lupus nephritis.

Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Although treatment with corticosteroids and cytotoxic drugs may be useful in many cases, morbidity associated with these drugs and the relapsing nature of the disease make it necessary to develop new treatment strategies. Five-month old female NZB/W F1 mice were divided into the following groups: CYP group (n = 10), cyclophosphamide (CYP) 50 mg/kg intraperitoneally every 10 days; RAPA 1 group (n = 10) oral daily sirolimus (SRL), 1 mg/kg; RAPA 12 group (n = 13), oral daily SRL, 12mg/kg; FTY group (n = 10), oral fingolimod (FTY720), 2 mg/kg three times per week. An additional group of 13 non-treated mice were used as a control (control group). Follow-up was performed over four months. Animal survival, body weight, anti-DNA antibodies and proteinuria were determined. Kidneys were processed for conventional histology and immunofluorescence for IgG and complement. Total histological score (HS) was the sum of mesangial expansion, endocapillary proliferation glomerular deposits, extracapillary proliferation, interstitial infiltrates, tubular atrophy and interstitial fibrosis. All treated groups had lower proteinuria at the end of the follow-up with respect to the control group (P < 0.0001). Serum anti-DNA antibodies were appropriately controlled in RAPA 1 and CYP groups, but not in FTY or RAPA 12 groups. SRL and CYP arrested, and perhaps reversed almost all histological lesions. FTY720 ameliorated histological lesions but did not control mesangial expansion or interstitial infiltrates. SRL produces great improvement in murine lupus nephritis, while FTY720 seems a promising alternative if used in appropriate doses.

The influence of local instillation of fusidic acid on the development of microbial complications after lung resection.

The efficacy of local instillation of fusidic acid in the prevention of post-surgical microbial complications during various types of lung resection was studied. Four hundred ninety two consecutive patients who underwent 504 thoracotomies for non-small cell lung carcinoma during April 1998-May 2004 were reviewed. The 290 patients of the first period who underwent 298 thoracotomies received a chemoprophylactic regimen of intravenous cefuroxime while the 202 patients of the second period who underwent 206 thoracotomies were additionally treated with fusidic acid, irrigated with local instillation into the pleural space, for the prevention of postoperative septic complications. Patients were followed postoperatively for development of septic complications (empyema and bronchopleural fistula) as well as of pneumonia and wound infection. Seventeen patients (5.7%) of the first period developed empyema and 13 fistula (4.4%), whereas only 2 patients (1.0%) of the second period developed empyema and fistula (OR = 5.876; 95% CI, 1.343- 25.716; P = 0.008 and OR = 4.193; 95% CI, 1.003-20.130; P = 0.034, respectively). Cases of pneumonia decreased, but not significantly, from 21 (7.0%) during the first period to 9 (4.4%) during the second period (OR = 1.613; 95% CI, 0.724-3.593; P = 0.257) while cases of wound infection decreased significantly from 19 (6.4%) to 2 (1.0%) (OR = 6.567; 95% CI, 1.513-28.510; P = 0.003). During the first period 23 pathogens were found from cases of empyema and 73 pathogens from cases of pneumonia and wound infection, whereas during the second period 3 and 18 pathogens were respectively found (OR = 5.3; 95% CI, 1.570-17.888; P = 0.003, and OR = 2.804; 95% CI, 1.628-4.838; P <0.001, respectively). These results indicate that local instillation of fusidic acid in the pleural space prior to lung resection seems effective in reducing the rate of septic complications as well as of wound infections.

Lung cancer: quality of life after surgery.

PURPOSE: Lung cancer is the most common cause of cancer death in both men and women in our country. It has been estimated that there will be 6,000 lung cancer deaths every year in Greece. However, many patients with bronchogenic carcinoma also have coexistent obstructive lung disease. In these patients, preoperative prediction of functional status after lung resection is mandatory. The aim of our study was to determine the effect of lung resection on postoperative spirometric lung function. PATIENTS AND METHODS: 112 patients underwent spirometric pulmonary tests preoperatively, and at 3 and 6 months after their operation. The predicted postoperative forced vital capacity (FVC) and forced expiratory volume in the first second (FEV1) were calculated using the formula of Juhl and Frost: predicted postoperative FEV1 (or FVC)=preoperative FEV1(or FVC) x[1-(S x 0.0526)], where S=number of segments resected. Statistical significance was defined as a p value < 0.05. RESULTS: The functional percentage losses at 6 months for lobectomies and pneumonectomies were 7.34% and 34.89% for FVC and 7.72%; and 32.53% for FEV, respectively. The linear regression analysis derived from the correlation between predicted and measured FEV1 resulted in 2 equations for lobectomy and pneumonectomy. The first, for lobectomy, was: FEV1POSTOP=0.00211 + 0.896660 x FEV1PREOP; and the second, for pneumonectomy, was: FEV1POSTOP=0.145 + 0.65318 x FEV1PREOP. CONCLUSION: We conclude that our formulas are a reliable method for predicting postoperative respiratory function of the patients with lung cancer.

Expandable wallstents for treatment of tracheoesophageal fistulas of malignant origin.

PURPOSE: To present our experience with endoscopic placement of esophageal endoprosthesis with self-expandable wallstents in patients with malignant tracheoesophageal fistulas. PATIENTS AND METHODS: 16 patients were retrospectively evaluated, in whom 16 stents were positioned at the esophagus because of tracheoesophageal fistulas: 12 of them suffered of malignant tumors of the esophagus and 4 of malignant tumors of the lung. All stents were placed with guide wire. We used self-expandable wallstents with internal silicon-basedcovering with flared ends, made of a stainless-steel alloy woven into a tubular mesh. RESULTS: Stents were successfully places in all patients. No procedure-related mortality or significant morbidity occured. Two patients complained of transient swallowing discomfort, but none of them required any additional analgesia. Thirty-day mortality was nil. Immediate leak occlusion was obtained on erect contrast assessment after the procedure in all patients. CONCLUSION: Self-expandable wallstents endoprosthesis in the esophagus for fistulas of malignant origin is an easy, well tolerated, safe and effective procedure without important complications or mortality.

Is there a role of whole body bone scan in early stages of non small cell lung cancer patients.

PURPOSE: The aim of our study was to re-evaluate the role of whole-body bone scanning (WBBS) in detecting bone metastases in apparently operable stages of non small cell lung cancer (NSCLC) patients. PATIENTS AND METHODS: We made a retrospective analysis of 60 patients (53 males, 7 females, aged 47-87 years, mean 68+/-4) between 2004-2006. All patients had a full series of imaging staging procedures including WBBS. Their medical records were reviewed with respect to how often bone metastases were detected and whether or not the patients showed any symptoms or laboratory abnormalities indicating bone involvement. RESULTS: Skeletal metastases (confirmed afterwards by x-ray, computed tomography or biopsy) were found in 11 (18.3%) patients. All of them had normal serum alkaline phosphatase and calcium concentrations. Eleven patients had symptoms suggesting bone metastases and 49 were asymptomatic. Bone metastases were detected in 3 (27.2%) of 11 clinically symptom-positive patients and in 8 (16.3%) of 49 clinically symptom-negative patients. CONCLUSION: The present study indicates that if bones scans were done only in patients reporting skeletal symptoms an important number of patients (16.3%) would have been misstaged due to asymptomatic bone metastases. We conclude that in patients with apparently operable NSCLC preoperative staging using WBBS is useful to avoid under-staging and futile surgery.

Diversidad y abundancia de equinodermos en la laguna arrecifal del Parque Nacional Cahuita, Caribe de Costa Rica / Diversity and abundance of echinoderms from the reef lagoon at Cahuita National Park, Caribbean from Costa Rica

A total of 15 species of echinoderms (one asteroid, seven ophiuroids, five echinoids and two holothurians) were recorded at the Cahuita National Park reef lagoon, between September and October 2003, using a 1 m2 quadrant. The sites with coral substrate and algae were the most diverse, while those with seagrass and sand were the least. Ophiuroids were the most abundant group with 170 individuals, asteroids were the least abundant. Adding other studies and reports of echinoderms to this study, a total of 23 species have been found at Cahuita National Park, which is the most diverse area on the Caribbean of Costa Rica. We report nine new echinoderm records for Costa Rica's Caribbean.

Direct electrotransfer of hHGF gene into kidney ameliorates ischemic acute renal failure.

In the early phase of kidney transplantation, the transplanted kidney is exposed to insults like ischemia/reperfusion, which is a leading cause of acute renal failure (ARF). ARF in the context of renal transplantation predisposes the graft to developing chronic damage and to long-term graft loss. Hepatocyte growth factor (HGF) has been suggested to support the intrinsic ability of the kidney to regenerate in response to injury by its morphogenic, mitogenic, motogenic and antiapoptotic activities. In the present paper, we examine whether human HGF (hHGF) gene electrotransfer helps in the recovery from ARF in a model of rat renal warm ischemia. We also assess the advantages of this form of gene therapy by direct electroporation of the kidney, given that transplantation offers the possibility of manipulating the organ in vivo. We have compared the therapeutic efficiency of two electroporation methodologies in a rat ARF model. Although they both targeted the same organ, the two methods were applied to different parts of the animal: muscle and kidney. Kidney direct electrotransfer was shown to be more efficient not only in pharmacokinetic but also in therapeutic terms, so it may become a clinically practical alternative in renal transplantation.

Percutaneous stent placement in malignant cases of superior vena cava syndrome.

PURPOSE: Superior vena cava (SVC) syndrome is caused by SVC stenosis or occlusion, frequently as a consequence of lung cancer or a mediastinal tumor. SVC syndrome is characterized by unpleasant symptoms and the condition usually leads to death if untreated. Treatment with radiation therapy and chemotherapy may produce an initial relief, whereas operations with bypass are associated with high mortality and morbidity. The PURPOSE of our study was to show the efficiency of percutaneous stenting in the SVC for relieving SVC syndrome secondary to malignant diseases. PATIENTS AND METHODS: From January 1999 to March 2003, 17 patients with malignant SVC syndrome were evaluated at the "Metaxa" Cancer Hospital. Their caval stenoses were confirmed by means of computed tomography and venography. There were 15 males and 2 females with a median age of 62 years (range 47-79). The SCV syndrome was caused by malignant disease in all patients: bronchogenic carcinoma in 14 and lymphoma in 3. All patients underwent placement of a self-expandable (wallstent) endovascular (vena cava) prosthesis. RESULTS: All procedures were successfully carried out without complications. The average time for wallstent placement was 37 min. There was no sign of bleeding and the wallstent was well positioned on chest roentgenograms. All patients, without exception, noticed an immediate improvement, with relief of dyspnea and rapid resolution of headache. Cyanosis disappeared over the first hour and swelling resolved gradually over the first 24 hours. CONCLUSION: Percutaneous venous wallstent placement in the SVC is a simple, safe and effective technique to rapidly relieve SVC syndrome caused by malignant diseases.

Surgery in small cell lung cancer: when and why.

Small cell lung cancer (SCLC) is considered a systemic disease at diagnosis, because the potential for hematogenous and lymphogenic metastases is very high. For many years, the diagnosis of SCLC was considered a contraindication for surgery because radiotherapy was at least equivalent in terms of local control, and the rate of resectability in SCLC patients was poor. When chemotherapy became the mainstay of treatment for SCLC, radiotherapy was its logical complement, and surgery was progressively abandoned. However, some centers continued to support surgery because experience suggested that in selected patients it was possible to achieve a long-term survival. In the search for predictors of long-term survival it became evident that the TNM staging system was effective for SCLC. The rationale for surgery in the context of SCLC is based on 3 factors: a) Several historical series of patients operated for limited-stage SCLC reported some long-term survivors, showing that cure could be achieved. b) After chemotherapy and radiotherapy, the rate of local relapse is 20%-30%. The assumption that surgical resection might be superior for local disease control has been suggested but not yet proved. c) The surgical intervention can precisely assess pathological (p) response to chemotherapy, identify carcinoids erroneously diagnosed as SCLC, and treat the non-small cell lung cancer (NSCLC) component of tumors with a mixed histology. Even if some controversies exist, it is accepted that surgery can be proposed as the first treatment in patients with T1 or T2 lesions with no evidence of lymph node involvement, followed by adjuvant chemotherapy. In more advanced stages of disease, chemotherapy should be the first step of treatment and surgery can be proposed to responding patients, before radical radiotherapy, depending on the p-stage of disease. Such an intensive multidisciplinary approach should be always employed in the context of controlled clinical trials.

Stimulation of innate immunity by susceptible and multidrug-resistant Pseudomonas aeruginosa: an in vitro and in vivo study.

In attempt to investigate the stimulatory effect of Pseudomonas aeruginosa on innate immunity and to correlate it to its level of resistance to antimicrobials, 20 isolates were applied; 8 isolates were susceptible and 12 multidrug-resistant. Genetic diversity was defined by PFGE. Human monocytes of two healthy volunteers were in vitro stimulated by the isolates for the production of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12) and anti-inflammatory cytokines (IL-10), of malondialdehyde and of procalcitonin. Cytokines were estimated by EIA, malondialdehyde by the thiobarbiturate assay and procalcitonin by an immunochemiluminometric assay. Survival of 48 Wistar rats was recorded after induction of sepsis by the intraperitoneal injection of three susceptible and three multidrug-resistant isolates. To test whether comparative effect of the latter isolates on survival correlates with any difference of monocyte-mediated release of pro-inflammatory mediators, monocytes of two rats were in vitro stimulated for the production of TNF-alpha and of malondialdehyde. In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL-1beta and of IL-6 compared to stimulation by multidrug-resistant isolates. Similar differences were found for TNF-alpha and IL-8, but they were not statistically significant. Production of IL-10 and IL-12 was not detected after stimulation with any isolate. Levels of procalcitonin were similar after induction with either susceptible or multidrug-resistant isolates. Mean survival of animals was 7.56, 21.80 and 55.20 h, respectively, after challenge by the susceptible isolates and 28.89, 61.8 and more than 120 h, respectively, after challenge by the multidrug-resistant isolates. Differences of survival were accompanied by greater rodent monocyte-release of TNF-alpha and malondialdehyde after stimulation by the susceptible isolates compared to multidrug-resistant ones. It is concluded that considerable differences are encountered on the stimulation of human monocytes by susceptible and resistant isolates of Pseudomonas aeruginosa. These results correlate with in vivo evidence and might influence decision on therapeutics.

Syndrome after pneumonectomy.

The syndrome after pneumonectomy is an unusual complication of pneumonectomy resulting from excessive displacement of the mediastinal structures toward the empty pleural space. In infants, children, and infrequently in young or middle- aged adults, excessive mediastinal shift after pneumonectomy occurs gradually. The resultant compression of the remaining contralateral bronchus leads to severe respiratory compromise. Development of the syndrome is most common after right pneumonectomy, is infrequently seen after left pneumonectomy in patients with a right-sided aortic arch, and is observed rarely after left pneumonectomy in patients with a normal position of the aortic arch in the left hemithorax. The typical clinical presentation is that of dyspnea, which occurs months or years after surgery, and the diagnosis is confirmed by documenting, on computed tomographic scan or by bronchoscopy, significant tracheobronchial obstruction. The problem can be corrected by restoration of the normal relation of the mediastinal structures, which is best achieved by inserting a tissue expander in the empty chest cavity. Endoluminal stenting seems to offer the most efficient treatment of associated tracheobronchomalacia.

Thoracoscopy, bronchoscopy and mediastinoscopy in the staging of lung cancer.

PURPOSE: To estimate the benefit of Video-Assisted Thoracoscopy (VAT) in the staging of patients with lung cancer. PATIENTS AND METHODS: Between October 1998 and January 2001 VAT was used in 250 patients with histologically proven lung cancer. They were staged by more conventional techniques including magnetic resonance imaging (MRI), bronchoscopy, and mediastinoscopy. RESULTS: As a result of VAT 30 patients were upstaged and spared a thoracotomy receiving neo-adjuvant chemotherapy at that point. In 40 patients the procedure was converted into an open thoracotomy and a curative resection was performed during the same session. The remaining 180 patients were deemed inoperable and they received chemotherapy and radiation treatment. CONCLUSION: VAT enhanced our bronchoscopic and medistinoscopic findings. It was especially useful in assessing the extent of invasion of various thoracic structures amenable to surgical removal "en block" with the tumor, and in differentiating simple contact of the tumor with an intrathoracic structure from tumor invasion. In addition, it allowed access and sampling of lymph nodes in spaces not easily accessible by mediastinoscopy.

Completion pneumonectomy for lung cancer.

PURPOSE: Completion pneumonectomy is a trully challenging operation associated with increased mortality and morbidity. The aim of this study was to present a series of 18 patients who underwent completion pneumonectomy for lung cancer during a 15-year period and to evaluate the postoperative outcomes and long-term results. PATIENTS AND METHODS: Between January 1985 and December 2000,18 patients underwent completion pneumonectomy for lung cancer; 10 for local recurrence ,6 for second primary lung tumor and 2 for lung tumors in patients who had previously been operated on for benign disease. RESULTS: No intraoperative deaths occurred. Postoperative mortality and morbidity were 11.11% and 33.33%, respectively. The median operational time was 212.7 minutes. The mean blood loss during the procedure was 1.042,5 ml. The complication rate was 33.33%. The 5-year survival was 18.75% for all patients. The 5-year survival was 25% for the local recurrence group and 50% for the primary lung cancer group. The 5-year survival of the patients in the second primary tumor group has not been reached yet. CONCLUSION: Completion pneumonectomy can be performed with an acceptable operative mortality rate and offers a second chance for cure to patients with lung cancer. Although complications are common ,they can successfully be managed with proper understanding of them.

Incisional metastasis after cervical mediastinoscopy: a case report.

The risk of iatrogenic tumor seeding from mediastinoscopy is low. The etiology of this complication remains unclear. We present the case of a patient with this condition, discuss the cause and management, and review the literature.