Post-Transplant Nephroptosis as a Cause of Recurrent Urinary Tract Infections: A Case Report.

BACKGROUND: The aim of this study was to present a rare cause of recurrent urinary tract infections (UTIs) in a patient after kidney transplantation. METHODS: The patient's consent was obtained, and full medical documentation was reviewed. After analyzing the literature, only 3 case reports of post-transplant nephroptosis were found. RESULTS: A 32-year-old woman with a history of type 1 diabetes, after kidney and pancreas transplantation a year earlier, was admitted to the hospital due to another incident of fever, dysuria, and pain in the lower abdomen. UTIs had been recurring for several months despite prophylaxis, initially with co-trimoxazole and then with fosfomycin. There were no anatomic abnormalities, and tacrolimus concentrations always remained at the lower range of normal. Kinking of the ureter was suspected because of a change in the position of the transplanted kidney. Ultrasonography performed in the standing and lying positions confirmed the diagnosis. A double J catheter was inserted into the ureter. In the following months, no UTI or urinary retention recurrence was observed. CONCLUSIONS: Nephroptosis of a transplanted kidney is extremely rare. The standard place for graft implantation-the iliac fossa-significantly limits the potential for migration. Kidneys implanted intraperitoneally also do not show clinically significant mobility due to postoperative adhesions. Floating kidneys potentially lead to serious complications. In addition to pain, a migrating graft may cause urine retention, predisposing to UTI and acute kidney injury.

On Whether Ca-125 Is the Answer for Diagnosing Overhydration, Particularly in End-Stage Kidney Disease Patients-A Systematic Review.

Overhydration (OH) is a prevalent medical problem that occurs in patients with kidney failure, but a specific marker has still not been found. Patients requiring kidney replacement therapy suffer from a water imbalance, which is correlated with mortality rates in this population. Currently, clinicians employ techniques such as bioimpedance spectroscopy (BIS) and ultrasound (USG) markers of overhydration or markers of heart and kidney function, namely NT-pro-BNP, GFR, or creatinine levels. New serum markers, including but not limited to Ca-125, galectin-3 (Gal-3), adrenomedullin (AMD), and urocortin-2 (UCN-2), are presently under research and have displayed promising results. Ca-125, which is a protein mainly used in ovarian cancer diagnoses, holds great potential to become an OH marker. It is currently being investigated by cardiologists as it corresponds to the volume status in heart failure (HF) and ventricular hypertrophy, which are also associated with OH. The need to ascertain a more precise marker of overhydration is urgent mainly because physical examinations are exceptionally inaccurate. The signs and symptoms of overhydration, such as edema or a gradual increase in body mass, are not always present, notably in patients with chronic kidney disease. Metabolic disruptions and cachexia can give a false picture of the hydration status. This review paper summarizes the existing knowledge on the assessment of a patient's hydration status, focusing specifically on kidney diseases and the role of Ca-125.

Fabry disease - what a gastroenterologist should know.

Fabry disease is a rare, X-linked metabolic error caused by various mutations in the α-galactosidase A gene, which results in the accumulation of glycosphingolipids. Gastrointestinal symptoms are quite common in affected patients; therefore, it is important for gastroenterologists to keep it in mind as a differential diagnosis for especially challenging patients. The following review provides concise information on epidemiology and genetics, signs, and symptoms of the disease, focusing on the gastrointestinal (GI) tract, providing a brief overview of the diagnostic process and the available treatment, both disease specific and supportive, again with a focus on alleviation of gastrointestinal symptoms.

Factors Influencing Longevity of Humoral Response to SARS-CoV-2 Vaccination in Patients with End Stage Kidney Disease Receiving Renal Replacement Therapy.

COVID-19 has severely affected the population of patients with end stage renal disease. Current data have proved a two-dose vaccination schedule against SARS-CoV-2 to be effective among dialyzed patients. There are limited data on the longevity and modulating factors of humoral response after vaccination. We performed a prospective longitudinal cohort study to determine longevity of the humoral response after SARS-CoV-2 vaccine. The study included 191 adult patients on hemodialysis and peritoneal dialysis. All participants had been vaccinated with three doses, either with BNT162b2 (Pfizer-BioNTech) (n = 109) or mRNA-1273 (Moderna) (n = 82). Anti-spike protein receptor-binding domain antibodies (anti-S IgG) were assessed using SARS-CoV-2 (RBD) IgG ELISA EIA-6150 IVD assay at baseline, on the 21st day and 43rd day, before a booster dose and two weeks thereafter. We found that before vaccination, 37.7% of the cohort had anti-S IgG titres concordant with seroconversion. After two-dose vaccination, seroconversion occurred in 97% of patients. The booster dose evoked a ~12-fold increase in antibody level. Obesity increased more than two-fold the odds for a decrease in anti-S IgG. Previous COVID-19 infection enhanced longevity of the humoral response following vaccination. In patients with previous COVID-19 infection, the BNT162b2 vaccine was associated with a higher odds of anti-S IgG waning compared to the mRNA-1273 vaccine. In conclusion, we report that obesity predisposes patients to protective antibody waning, hybrid immunity enhances odds for higher anti-S IgG concentrations and vaccine efficacy may be influenced by previous SARS-CoV-2 infection. The results might provide a rationale for vaccination protocol design.

The Impact of Cardiovascular Risk Factors on the Course of COVID-19.

AIM OF THE STUDY: The aim of our review is to indicate and discuss the impact of cardiovascular risk factors, such as obesity, diabetes, lipid profile, hypertension and smoking on the course and mortality of COVID-19 infection. BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is spreading around the world and becoming a major public health crisis. All coronaviruses are known to affect the cardiovascular system. There is a strong correlation between cardiovascular risk factors and severe clinical complications, including death in COVID-19 patients. All the above-mentioned risk factors are widespread and constitute a significant worldwide health problem. Some of them are modifiable and the awareness of their connection with the COVID-19 progress may have a crucial impact on the current and possible upcoming infection. DATA COLLECTION: We searched for research papers describing the impact of selected cardiovascular risk factors on the course, severity, complications and mortality of COVID-19 infection form PubMed and Google Scholar databases. Using terms, for example: "COVID-19 cardiovascular disease mortality", "COVID-19 hypertension/diabetes mellitus/obesity/dyslipidemia", "cardiovascular risk factors COVID-19 mortality" and other related terms listed in each subtitle. The publications were selected according to the time of their publications between January 2020 and December 2021. From the PubMed database we obtain 1552 results. Further studies were sought by manually searching reference lists of the relevant articles. Relevant articles were selected based on their title, abstract or full text. Articles were excluded if they were clearly related to another subject matter or were not published in English. The types of articles are mainly randomized controlled trial and systematic review. An additional criterion used by researchers was co-morbidities and age of patients in study groups. From a review of the publications, 105 of them were selected for this work with all subheadings included. Findings and Results: The intention of this review was to summarize current knowledge about comorbidities and development of COVID-19 infection. We tried to focus on the course and mortality of the abovementioned virus disease in patients with concomitant CV risk factors. Unfortunately, we were unable to assess the quality of data in screened papers and studies we choose because of the heterogenicity of the groups. The conducted studies had different endpoints and included different groups of patients in terms of nationality, age, race and clinical status. We decide to divide the main subjects of the research into separately described subtitles such as obesity, lipid profile, hypertension, diabetes, smoking. We believe that the studies we included and gathered are very interesting and show modern and present-day clinical data and approaches to COVID-19 infection in specific divisions of patients.

IGFBP7 Concentration May Reflect Subclinical Myocardial Damage and Kidney Function in Patients with Stable Ischemic Heart Disease.

The objective of this study was to determine the associations between insulin-like growth-factor-binding protein 7(IGFBP7) concentrations and concentrations of troponin T(TnT), N-terminal pro-B-type natriuretic peptide(NT-proBNP) and the parameters of kidney function in patients with stable ischemic heart disease(IHD). The IHD group consisted of 88 patients, and the population group comprised 66 subjects without a history of IHD. IGFBP7, TnT and NTproBNP concentrations were measured. The IGFBP7 value was considerably higher in the IHD group (1.76 ± 1 ng/mL vs. 1.43 ± 0.44 ng/mL, respectively, p = 0.019). Additionally, IHD subjects had a significantly higher concentration of TnT and NTproBNP. In both groups there was a significant correlation between IGFBP7 and serum parameters of kidney function (creatinine concentration: population gr. r = 0.45, p < 0.001, IHD gr. r = 0.86, p < 0.0001; urea concentration: population gr. r = 0.51, p < 0.0001, IHD gr. r = 0.71, p < 0.00001). No correlation between IGFBP7 and microalbuminuria or the albumin to creatinine ratio in urine was found. Moreover, there was a significant correlation between IGFBP7 concentration and markers of heart injury/overload-TnT and NT-BNP(r = 0.76, p < 0.001 and r = 0.72, p < 0.001, respectively). Multivariate regression analysis in joint both revealed that the IGFBP7 concentration is independently associated with urea, creatinine and TnT concentrations (R2 for the model 0.76). IHD patients presented significantly higher IGFBP7 concentrations than the population group. Elevated IGFBP7 levels are associated predominantly with markers of kidney function and myocardial damage or overload.

Water - A life-giving toxin - A nephrological oxymoron. Health consequences of water and sodium balance disorders. A review article.

BACKGROUND: This article aims to reveal misconceptions about methods of assessment of hydration status and impact of the water disorders on the progression of kidney disease or renal dysfunction. MATERIALS AND METHODS: The PubMed database was searched for reviews, meta-analyses and original articles on hydration, volume depletion, fluid overload and diagnostic methods of hydration status, which were published in English. RESULTS: Based on the results of available literature the relationship between the amount of fluid consumed, and the rate of progression of chronic kidney disease, autosomal dominant polycystic kidney disease, and kidney stones disease was discussed. Selected aspects of the assessment of the hydration level in clinical practice based on physical examination, laboratory tests, and imaging are presented. The subject of in-hospital fluid therapy is discussed. Based on available randomized studies, an attempt was made to assess, which fluids should be selected for intravenous treatment. CONCLUSIONS: There is some evidence for the beneficial effect of increased water intake in preventing recurrent cystitis and kidney stones, but there are still no convincing data for chronic kidney disease and autosomal dominant polycystic kidney disease. Further studies are needed to clarify the aforementioned issues and establish a reliable way to assess the volemia and perform suitable fluid therapy.

Show Me What You Have Inside-The Complex Interplay between SIBO and Multiple Medical Conditions-A Systematic Review.

The microbiota, as a complex of microorganisms in a particular ecosystem, is part of the wider term-microbiome, which is defined as the set of all genetic content in the microbial community. Imbalanced gut microbiota has a great impact on the homeostasis of the organism. Dysbiosis, as a disturbance in bacterial balance, might trigger or exacerbate the course of different pathologies. Small intestinal bacterial overgrowth (SIBO) is a disorder characterized by differences in quantity, quality, and location of the small intestine microbiota. SIBO underlies symptoms associated with functional gastrointestinal disorders (FGD) as well as may alter the presentation of chronic diseases such as heart failure, diabetes, etc. In recent years there has been growing interest in the influence of SIBO and its impact on the whole human body as well as individual systems. Therefore, we aimed to investigate the co-existence of SIBO with different medical conditions. The PubMed database was searched up to July 2022 and we found 580 original studies; inclusion and exclusion criteria let us identify 112 eligible articles, which are quoted in this paper. The present SIBO diagnostic methods could be divided into two groups-invasive, the gold standard-small intestine aspirate culture, and non-invasive, breath tests (BT). Over the years scientists have explored SIBO and its associations with other diseases. Its role has been confirmed not only in gastroenterology but also in cardiology, endocrinology, neurology, rheumatology, and nephrology. Antibiotic therapy could reduce SIBO occurrence resulting not only in the relief of FGD symptoms but also manifestations of comorbid diseases. Although more research is needed, the link between SIBO and other diseases is an important pathway for scientists to follow.

Plasmid Mediated mcr-1.1 Colistin-Resistance in Clinical Extraintestinal Escherichia coli Strains Isolated in Poland.

Objectives: The growing incidence of multidrug-resistant (MDR) bacteria is an inexorable and fatal challenge in modern medicine. Colistin is a cationic polypeptide considered a "last-resort" antimicrobial for treating infections caused by MDR Gram-negative bacterial pathogens. Plasmid-borne mcr colistin resistance emerged recently, and could potentially lead to essentially untreatable infections, particularly in hospital and veterinary (livestock farming) settings. In this study, we sought to establish the molecular basis of colistin-resistance in six extraintestinal Escherichia coli strains. Methods: Molecular investigation of colistin-resistance was performed in six extraintestinal E. coli strains isolated from patients hospitalized in Medical University Hospital, Bialystok, Poland. Complete structures of bacterial chromosomes and plasmids were recovered with use of both short- and long-read sequencing technologies and Unicycler hybrid assembly. Moreover, an electrotransformation assay was performed in order to confirm IncX4 plasmid influence on colistin-resistance phenotype in clinical E. coli strains. Results: Here we report on the emergence of six mcr-1.1-producing extraintestinal E. coli isolates with a number of virulence factors. Mobile pEtN transferase-encoding gene, mcr-1.1, has been proved to be encoded within a type IV secretion system (T4SS)-containing 33.3 kbp IncX4 plasmid pMUB-MCR, next to the PAP2-like membrane-associated lipid phosphatase gene. Conclusion: IncX4 mcr-containing plasmids are reported as increasingly disseminated among E. coli isolates, making it an "epidemic" plasmid, responsible for (i) dissemination of colistin-resistance determinants between different E. coli clones, and (ii) circulation between environmental, industrial, and clinical settings. Great effort needs to be taken to avoid further dissemination of plasmid-mediated colistin resistance among clinically relevant Gram-negative bacterial pathogens.

Locked Away-Prophylaxis and Management of Catheter Related Thrombosis in Hemodialysis.

Reliable vascular access is necessary for effective hemodialysis. Guidelines recommend chronic hemodialysis via an arteriovenous fistula (AVF), however, in a significant number of patients, permanent central venous catheters (CVCs) are used. The use of a tunneled catheter is acceptable if the estimated dialysis time is less than a year or it is not possible to create an AVF. The main complications associated with CVC include thrombosis and catheter-related bloodstream infections (CRBSIs), which may result in loss of vascular access. The common practice is to use locking solutions to maintain catheter patency and minimize the risk of CRBSI. This paperwork summarizes information on currently available locking solutions for dialysis catheters along with their effectiveness in preventing thrombotic and infectious complications and describes methods of dealing with catheter dysfunction. The PubMed database was systematically searched for articles about locking solutions used in permanent CVCs in hemodialysis patients. Additional studies were identified by searching bibliographies and international guidelines. Articles on end-stage kidney disease patients dialyzed through a permanent CVC were included. Information from each primary study was extracted using pre-determined criteria including thrombotic and infectious complications of CVC use, focusing on permanent CVC if sufficient data were available. Of the currently available substances, it seems that citrate at a concentration of 4% has the best cost-effectiveness and safety profile, which is reflected in the international guidelines. Recent studies suggest the advantage of 2+1 protocols, i.e., taurolidine-based solutions with addition of urokinase once a week, although it needs to be confirmed by further research. Regardless of the type of locking solution, if prophylaxis with a thrombolytic agent is chosen, it should be started from the very beginning to reduce the risk of thrombotic complications. In case of CVC dysfunction, irrespective of the thrombolysis attempt, catheter replacement should be planned as soon as possible.

[Current nutrition recommendations for chronic kidney disease]. / Aktualne zalecenia zywienia w przewleklej chorobie nerek.

The paper discusses the current recommendations regarding the supply of individual nutrients in patients with chronic kidney disease (CKD). The recommendations include keeping the energy supply in the range of 25 to 35 kcal per kilogram of proper body weight per day, limiting the consumption of phosphorus to maximum of 1 g per day and limiting sodium to maximum of 2.3 g per day. In patients with eGFR <30 ml / min / 1.73 m2, a potassium restriction should be added so that its concentration in the blood does not exceed 5 mmol / l. Experts' views on the protein restriction in CKD patients are divided. The topic is controversial and more researches are needed to see if reducing protein intake leads to malnutrition and increased risk of death in this population. The results of studies on the use of a vegetarian diet in patients with CKD seem to be promising. It is good to remember about consuming appropriate amounts of products containing trace elements such as zinc, selenium or copper, as well as polyphenols, flavonoids and antioxidants.

Sirtuin 1 and Skin: Implications in Intrinsic and Extrinsic Aging-A Systematic Review.

Skin, as the outermost organ of the body, is constantly exposed to both intrinsic and extrinsic causative factors of aging. Intrinsic aging is related to compromised cellular proliferative capacity, and may be accelerated by harmful environmental influences with the greatest significance of ultraviolet radiation exposure, contributing not only to premature aging, but also to skin carcinogenesis. The overall skin cancer burden and steadily increasing global antiaging market provide an incentive for searching novel targets to improve skin resistance against external injury. Sirtuin 1, initially linked to extension of yeast and rodent lifespan, plays a key role in epigenetic modification of proteins, histones, and chromatin by which regulates the expression of genes implicated in the oxidative stress response and apoptosis. The spectrum of cellular pathways regulated by sirtuin 1 suggests its beneficial impact on skin aging. However, the data on its role in carcinogenesis remains controversial. The aim of this review was to discuss the relevance of sirtuin 1 in skin aging, in the context of intrinsic factors, related to genetic premature aging syndromes, as well as extrinsic modifiable ones, with the assessment of its future application. PubMed were searched from inception to 4 January 2021 for relevant papers with further search carried out on ClinicalTrials.gov. The systematic review included 46 eligible original articles. The evidence from numerous studies proves sirtuin 1 significance in both chronological and premature aging as well as its dual role in cancer development. Several botanical compounds hold the potential to improve skin aging symptoms.

Serum sirtuin 1 is independently associated with intact PTH among patients with chronic kidney disease.

BACKGROUND: Sirtuin 1 is involved in the pathogenesis of age-related diseases. PURPOSE: The aim of the study was to assess the clinical and diagnostic value of serum sirtuin 1 concentration in patients with CKD. PATIENTS AND METHODS: The serum sirtuin 1 level was evaluated using ELISA kit in 100 CKD patients stratified for five stages and in a control group of 24 healthy volunteers. RESULTS: Serum sirtuin 1 concentration was higher in the CKD group compared with the control group (p<0.05). Sirtuin 1 correlated with conventional CKD biomarkers and eGFR equations, intact parathyroid hormone (iPTH) and age (all p<0.05). Statins, AT1 receptor antagonists and ß-blockers use were associated with decreased sirtuin concentration (p<0.05). Sirtuin 1 was able to distinguish CKD from control group with high sensitivity and specificity (93% and 87%, respectively; AUC=0.954). Surprisingly, after adjustment only iPTH concentration was an independent predictor of sirtuin 1 level. CONCLUSION: The association between sirtuin 1, eGFR equations and iPTH indicates its possible usefulness as a kidney function marker. In terms of iPTH being the only independent predictor of circulating sirtuin 1 it can be considered as an indirect cardiovascular risk biomarker regardless of renal function and provide additional information for patient management. Alternatively, sirtuin 1 is recognized as protective against vascular disease, and we demonstrated a positive correlation with iPTH, which may be related to accumulation of (7-84)-PTH having opposite biological effects to full-length PTH. Further studies are needed to explore the interplay between sirtuin 1, PTH and CKD-related vascular calcification as well as to assess its prognostic value in observational studies.

TRAIL and Cardiovascular Disease-A Risk Factor or Risk Marker: A Systematic Review.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic protein showing broad biological functions. Data from animal studies indicate that TRAIL may possibly contribute to the pathophysiology of cardiomyopathy, atherosclerosis, ischemic stroke and abdominal aortic aneurysm. It has been also suggested that TRAIL might be useful in cardiovascular risk stratification. This systematic review aimed to evaluate whether TRAIL is a risk factor or risk marker in cardiovascular diseases (CVDs) focusing on major adverse cardiovascular events. Two databases (PubMed and Cochrane Library) were searched until December 2020 without a year limit in accordance to the PRISMA guidelines. A total of 63 eligible original studies were identified and included in our systematic review. Studies suggest an important role of TRAIL in disorders such as heart failure, myocardial infarction, atrial fibrillation, ischemic stroke, peripheral artery disease, and pulmonary and gestational hypertension. Most evidence associates reduced TRAIL levels and increased TRAIL-R2 concentration with all-cause mortality in patients with CVDs. It is, however, unclear whether low TRAIL levels should be considered as a risk factor rather than a risk marker of CVDs. Further studies are needed to better define the association of TRAIL with cardiovascular diseases.

Mid-Regional Proadrenomedullin as a New Biomarker of Kidney and Cardiovascular Diseases-Is It the Future?

The increasing prevalence of cardiovascular disease and concomitant chronic kidney disease among the aging populations is responsible for considerable growth of mortality. Additionally, frequent, prolonged hospitalizations and long-term treatment generates progressive decline in bodily functions as well as substantial public health and economic burden. Accessibility to easy, non-invasive prognostic markers able to detect patients at risk of cardiovascular events may improve effective therapy and mitigate disease progression. Moreover, an early diagnosis allows time for implementation of prophylactic and educational programs that may result in decreased morbidity, improved quality of life and reduced public health expenditure. One of the promising candidates for a novel cardiovascular biomarker is mid-regional proadrenomedullin, a derivative of adrenomedullin. Adrenomedullin is a peptide hormone known for its vasodilatory, antioxidant, antiapoptotic and antifibrotic effects. A remarkable advantage of mid-regional proadrenomedullin is its longer half-life which is a prerequisite for plasma measurements. These review aims to discuss the importance of mid-regional proadrenomedullin with reference to its usefulness as a biomarker of increased cardiovascular risk and kidney disease progression.

The switch from proteasome to immunoproteasome is increased in circulating cells of patients with fast progressive immunoglobulin A nephropathy and associated with defective CD46 expression.

The proteasome to immunoproteasome (iPS) switch consists of ß1, ß2 and ß5 subunit replacement by low molecular weight protein 2 (LMP2), LMP7 and multicatalytic endopeptidase-like complex-1 (MECL1) subunits, resulting in a more efficient peptide preparation for major histocompatibility complex 1 (MHC-I) presentation. It is activated by toll-like receptor (TLR) agonists and interferons and may also be influenced by genetic variation. In a previous study we found an iPS upregulation in peripheral cells of patients with immunoglobulin A nephropathy (IgAN). We aimed to investigate in 157 IgAN patients enrolled through the multinational Validation Study of the Oxford Classification of IgAN (VALIGA) study the relationships between iPS switch and estimated glomerular filtration rate (eGFR) modifications from renal biopsy to sampling. Patients had a previous long follow-up (6.4 years in median) that allowed an accurate calculation of their slope of renal function decline. We also evaluated the effects of the PSMB8/PSMB9 locus (rs9357155) associated with IgAN in genome-wide association studies and the expression of messenger RNAs (mRNAs) encoding for TLRs and CD46, a C3 convertase inhibitor, acting also on T-regulatory cell promotion, found to have reduced expression in progressive IgAN. We detected an upregulation of LMP7/ß5 and LMP2/ß1 switches. We observed no genetic effect of rs9357155. TLR4 and TLR2 mRNAs were found to be significantly associated with iPS switches, particularly TLR4 and LMP7/ß5 (P < 0.0001). The LMP7/ß5 switch was significantly associated with the rate of eGFR loss (P = 0.026), but not with eGFR at biopsy. Fast progressors (defined as the loss of eGFR >75th centile, i.e. -1.91 mL/min/1.73 m2/year) were characterized by significantly elevated LMP7/ß5 mRNA (P = 0.04) and low CD46 mRNA expression (P < 0.01). A multivariate logistic regression model, categorizing patients by different levels of kidney disease progression, showed a high prediction value for the combination of high LMP7/ß5 and low CD46 expression.

The Links between Microbiome and Uremic Toxins in Acute Kidney Injury: Beyond Gut Feeling-A Systematic Review.

The last years have brought an abundance of data on the existence of a gut-kidney axis and the importance of microbiome in kidney injury. Data on kidney-gut crosstalk suggest the possibility that microbiota alter renal inflammation; we therefore aimed to answer questions about the role of microbiome and gut-derived toxins in acute kidney injury. PubMed and Cochrane Library were searched from inception to October 10, 2020 for relevant studies with an additional search performed on ClinicalTrials.gov. We identified 33 eligible articles and one ongoing trial (21 original studies and 12 reviews/commentaries), which were included in this systematic review. Experimental studies prove the existence of a kidney-gut axis, focusing on the role of gut-derived uremic toxins and providing concepts that modification of the microbiota composition may result in better AKI outcomes. Small interventional studies in animal models and in humans show promising results, therefore, microbiome-targeted therapy for AKI treatment might be a promising possibility.