Distinctive Olfactory Pattern in Parkinson's Disease and Non-Neurodegenerative Causes of Hyposmia.

BACKGROUND: Olfactory dysfunction is common in Parkinson's disease (PD). The characteristics of the hyposmia in PD have not been well defined. OBJECTIVE: To characterize the pattern of the olfactory deficit in PD and in other non-neurodegenerative aetiologies of hyposmia. METHODS: We evaluated 36 PD patients, 20 patients with hyposmia secondary to acute respiratory infection (ARI), and 19 patients with hyposmia secondary to traumatic brain injury (TBI). For comparison purposes, we included a group of 15 controls age and sex matched with PD patients. PD patients were classified based on disease duration and severity in de novo PD, and PD with and without chronic levodopa-related complications. The Barcelona Smell Identification Test was applied to all participants. RESULTS: For the first cranial nerve odours, PD patients scored lower than controls on smell detection (85.28 vs. 97.67%, p = 0.006), definition (79.58 vs. 93.33%, p = 0.007), recognition (63.33 vs. 81%, p = 0.020), and forced choice (58.06 vs. 82%, p < 0.001). Compared with ARI, forced choice was significantly better in PD patients (p < 0.001), but no differences were found regarding other olfactory characteristics. TBI patients showed significantly lower scores than the other study groups in all the olfaction items. For the fifth cranial nerve odours, recognition (p = 0.003) and identification (p = 0.019) were lower in the TBI group than in the others. No differences were found among PD subgroups regarding any olfactory characteristic. CONCLUSIONS: A differential pattern of hyposmia was observed in PD patients compared to other non-neurodegenerative aetiologies. Further studies with larger samples should replicate our results.

Electrochemical Genetic Profiling of Single Cancer Cells.

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present a simple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

Disulfide-modified antigen for detection of celiac disease-associated anti-tissue transglutaminase autoantibodies.

A simple and rapid immunosensor for the determination of the celiac disease-related antibody, anti-tissue transglutaminase, was investigated. The antigenic protein tissue transglutaminase was chemically modified, introducing disulfide groups through different moieties of the molecule (amine, carboxylic, and hydroxyl groups), self-assembled on gold surfaces, and used for the detection of IgA and IgG autoantibodies. The modified proteins were evaluated using enzyme-linked immunosorbent assay and surface plasmon resonance, which showed that only introduction of the disulfide groups through amine moieties in the tissue transglutaminase preserved its antigenic properties. The disulfide-modified antigen was co-immobilized via chemisorption with a poly(ethylene glycol) alkanethiol on gold electrodes. The modified electrodes were then exposed to IgA anti-tissue transglutaminase antibodies and subsequently to horseradish peroxidase-labeled anti-idiotypic antibodies, achieving a detection limit of 260 ng ml-1. Immunosensor performance in the presence of complex matrixes, including clinically relevant serum reference solutions and real patient samples, was evaluated. The introduction of disulfides in the antigenic protein enabled a simple and convenient one-step surface immobilization procedure involving only spontaneous gold-thiol covalent binding. Complete amperometric assay time was 30 min.

Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.

The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot.

[Traumatic brain injury in patients over 65 years of age attended in the emergency department, characteristics and complications]. / Traumatismo craneoencefálico en pacientes mayores de 65 años atendidos en urgencias; características y complicaciones.

BACKGROUND AND OBJECTIVE: Ageing is associated with an increased risk of falls and trauma. The aim of the study was to assess the characteristics of patients over 65 years of age who consulted the ED for traumatic brain injury (TBI) in 2022, their relationship with cognitive impairment, functional dependence, use of oral antiplatelet/anticoagulant drugs and complications. MATERIALS AND METHODS: Retrospective study conducted from 1 January to 31 December 2022. Demographic data were collected: age, sex, origin; cardiovascular risk factors; cognitive impairment using the Pfeiffer questionnaire; physical disability according to the Barthel Index; number of drugs; use of antiplatelet and oral anticoagulant (OAC); mechanism of fall; performance of cranial X-ray/CT, and presence of complications: intracranial haemorrhage (ICH), death. RESULTS: 599 patients were included. The mean age was 82.3±8.2 years. 63.8% were female and 36.2% male. 75.3% were from home, 24.7% from residence. No dementia in 61.4%, moderate-severe dementia in 38.6%. 58.1% were functionally independent, 25.1% had moderate-severe dependence. 85.7% had CVRF: HT 476 (79.5%), dyslipidaemia 354 (59.1%), DM 217 (36.2%), obesity 173 (28.9%), smoking 15 (2.5%). The number of drugs per patient was 9.2±4.3. Polypharmacy was present in 94.7% of patients. 35.9% were taking antiplatelet drugs and 30.2% anticoagulants. Intracranial haemorrhage occurred in 11 (2.3%) patients. Four (0.7%) patients died. CONCLUSIONS: The TBI in our study was caused by low-energy trauma in a female patient, without dementia, functionally independent and with polypharmacy. There were few serious complications: 2.3% ICH and 0.7% deaths. 90.1% of ICH occurred in patients on antiplatelet and/or OAC therapy.

Anatomic Feasibility of a Superficial Temporal Artery to Distal Anterior Cerebral Artery Bypass With a Parietal Branch Interposition Graft: The Superficial Temporal Artery Telescope Bypass.

BACKGROUND AND OBJECTIVES: Revascularizing the postcommunicating segment of the anterior cerebral artery (ACA) using extracranial donor sites requires long interposition grafts. The superficial temporal artery (STA) is frequently used for extracranial-intracranial ACA revascularization. However, the length of either STA branch is not sufficient to reach the ACA with a proper caliber match, so an interposition graft is required. The aim of this study was to evaluate a bypass that uses the 2 main branches of the STA to reach the A3 (pericallosal) segment of the ACA. METHODS: The frontal and parietal branches of the STA were dissected from 10 cadaveric specimens. The middle internal frontal artery (MIFA) was exposed through an anterior interhemispheric approach. An interposition graft technique was applied using the parietal branch of the STA (pSTA) to connect the frontal branch of the STA (fSTA) with the MIFA. The bypass code is fSTA (E-Ec) pSTA + pSTA (E-Sc) MIFA. Measurements of length and caliber were taken at the anastomotic sites for the distal branches of the STA and the MIFA. RESULTS: The mean (SD) diameter of the MIFA measured 1.4 (0.2) mm, similar to the calibers of the frontal and parietal branches of the STA. The mean (SD) length of the end-to-side STA-MIFA bypass was 145.5 (7.4) mm, and the mean (SD) length of the donor-graft construct measured 204.2 (27.9) mm. This bypass design resulted in a surplus donor graft length of 38%. CONCLUSION: Using the pSTA as an interposition graft proved to be a successful technique for creating an STA-MIFA bypass, yielding excess donor graft length that facilitated an unstrained bypass construct. This approach offers several advantages, including a single skin incision, ample graft length, caliber compatibility, and a straightforward technical execution.

Genomic Alterations in Molecularly Defined Oligodendrogliomas.

BACKGROUND AND OBJECTIVES: Oligodendrogliomas are defined by IDH1/2 mutation and codeletion of chromosome arms 1p/19q. Although previous studies identified CIC, FUBP1, and TERTp as frequently altered in oligodendrogliomas, the clinical relevance of these molecular signatures is unclear. Moreover, previous studies predominantly used research panels that are not readily available to providers and patients. Accordingly, we explore genomic alterations in molecularly defined oligodendrogliomas using clinically standardized next-generation sequencing (NGS) panels. METHODS: A retrospective single-center study evaluated adults with pathologically confirmed IDH-mutant, 1p/19q-codeleted oligodendrogliomas diagnosed between 2005 and 2021. Genetic data from formalin-fixed, paraffin-embedded specimens were analyzed with the NGS Solid Tumor Panel at the Johns Hopkins Medical Laboratories, which tests more than 400 cancer-related genes. Kaplan-Meier plots and log-rank tests compared progression-free survival (PFS) and overall survival by variant status. χ2 tests, t-tests, and Wilcoxon rank-sum tests were used to compare clinical characteristics between genomic variant status in the 10 most frequently altered genes. RESULTS: Two hundred and seventy-seven patients with molecularly defined oligodendrogliomas were identified, of which 95 patients had available NGS reports. Ten genes had 9 or more patients with a genomic alteration, with CIC, FUBP1, and TERTp being the most frequently altered genes (n = 60, 23, and 22, respectively). Kaplan-Meier curves showed that most genes were not associated with differences in PFS or overall survival. At earlier time points (PFS <100 months), CIC alterations conferred a reduction in PFS in patients (P = .038). CONCLUSION: Our study confirms the elevated frequency of CIC, FUBP1, and TERTp alterations in molecularly defined oligodendrogliomas and suggests a potential relationship of CIC alteration to PFS at earlier time points. Understanding these genomic variants may inform prognosis or therapeutic recommendations as NGS becomes routine.

Brain metastasis treatment guidelines: consensus by the Spanish Society of Neurosurgery Tumor Section.

Brain metastases are tumors that arise from a tumor cell originated in another organ reaching the brain through the blood. In the brain this tumor cell is capable of growing and invading neighboring tissues, such as the meninges and bone. In most patients a known tumor is present when the brain lesion is diagnosed, although it is possible that the first diagnose is the brain tumor before there is evidence of cancer elsewhere in the body. For this reason, the neurosurgeon must know the management that has shown the greatest benefit for brain metastasis patients, so treatments can be streamlined and optimized. Specifically, in this document, the following topics will be developed: selection of the cancer patient candidate for surgical resection and the role of the neurosurgeon in the multidisciplinary team, the importance of immunohistological and molecular diagnosis, surgical techniques, radiotherapy techniques, treatment updates of chemotherapy and immunotherapy and management algorithms in brain metastases. With this consensus manuscript, the tumor group of the Spanish Society of Neurosurgery (GT-SENEC) exposes the most relevant neurosurgical issues and the fundamental aspects to harmonize multidisciplinary treatment, especially with the medical specialties that are treating or will treat these patients.

Low grade gliomas guide-lines elaborated by the tumor section of Spanish Society of Neurosurgery.

Adult low-grade gliomas (Low Grade Gliomas, LGG) are tumors that originate from the glial cells of the brain and whose management involves great controversy, starting from the diagnosis, to the treatment and subsequent follow-up. For this reason, the Tumor Group of the Spanish Society of Neurosurgery (GT-SENEC) has held a consensus meeting, in which the most relevant neurosurgical issues have been discussed, reaching recommendations based on the best scientific evidence. In order to obtain the maximum benefit from these treatments, an individualised assessment of each patient should be made by a multidisciplinary team. Experts in each LGG treatment field have briefly described it based in their experience and the reviewed of the literature. Each area has been summarized and focused on the best published evidence. LGG have been surrounded by treatment controversy, although during the last years more accurate data has been published in order to reach treatment consensus. Neurosurgeons must know treatment options, indications and risks to participate actively in the decision making and to offer the best surgical treatment in every case.

Three-dimensional arrangement of short DNA oligonucleotides at surfaces via the synthesis of DNA-branched polyacrylamide brushes by SI-ATRP.

Short DNA oligonucleotide branches are incorporated into acrylamide brushes via surface initiated atom transfer radical polymerization in an attempt to increase DNA surface density by building three-dimensional molecular architectures. ATR-FTIR as well as hybridization studies followed by SPR confirm the incorporation of the DNA sequences into the polymer backbone. MALDI-TOF analysis further suggests that six acrylamide monomer units are typically separating DNA branches present on a single brushes approximately 26 units long. This new approach offers a promising alternative to SAM-based nucleic acid and aptamer sensors and could enable the realization of more complex soft materials of controlled architecture capable of both recognition and signaling by including additional optically or electrochemically active moieties.

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification.

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP-HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L(-1), with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.

Intraoperative imaging in the neurosurgery operating theatre: A review of the most commonly used techniques for brain tumour surgery. / Imagen intraoperatoria en el quirófano de neurocirugía: revisión de las técnicas más empleadas para la cirugía de los tumores cerebrales.

INTRODUCTION: New intraoperative imaging techniques, which aim to improve tumour resection, have been implemented in recent years in brain tumour surgery, although they lead to an increase in resources. In order to carry out an update on this topic, this manuscript has been drafted by a group from the Sociedad Española de Neurocirugía (Spanish Society of Neurosurgery). MATERIAL AND METHODS: Experts in the use of each one of the most-used intraoperative techniques in brain tumour surgery were presented with a description of the technique and a brief review of the literature. Indications for use, their advantages and disadvantages based on clinical experience and on what is published in the literature will be described. RESULTS: The most robust intraoperative imaging technique appears to be low- and high-field magnetic resonance imaging, but this is the technique which results in the greatest expenditure. Intraoperative ultrasound navigation is portable and less expensive, but it provides poorer differentiation of high-grade tumours and is observer-dependent. The most-used fluorescence techniques are 5-aminolevulinic acid for high-grade gliomas and fluorescein, useful in lesions which rupture the blood-brain barrier. Last of all, intraoperative CT is more versatile in the neurosurgery operating theatre, but it has fewer indications in neuro-oncology surgery. CONCLUSIONS: Intraoperative imaging techniques are used with increasingly greater frequency in brain tumour surgery, and the neurosurgeon should assess their possible use depending on their resources and the needs of each patient.

Glioblastoma treatment guidelines: Consensus by the Spanish Society of Neurosurgery Tumor Section. / Consenso sobre guías de tratamiento de los glioblastomas elaborado por el Grupo de Trabajo de Neurooncología (GTNO) de la SENEC.

INTRODUCTION: Glioblastoma (GBM) treatment starts in most patients with surgery, either resection surgery or biopsy, to reach a histology diagnose. Multidisciplinar team, including specialists in brain tumors diagnose and treatment, must make an individualize assessment to get the maximum benefit of the available treatments. MATERIAL AND METHODS: Experts in each GBM treatment field have briefly described it based in their experience and the reviewed of the literature. RESULTS: Each area has been summarized and the consensus of the brain tumor group has been included at the end. CONCLUSIONS: GBM are aggressive tumors with a dismal prognosis, however accurate treatments can improve overall survival and quality of life. Neurosurgeons must know treatment options, indications and risks to participate actively in the decision making and to offer the best surgical treatment in every case.

Design and testing of a packaged microfluidic cell for the multiplexed electrochemical detection of cancer markers.

We present the rapid prototyping of electrochemical sensor arrays integrated to microfluidics towards the fabrication of integrated microsystems prototypes for point-of-care diagnostics. Rapid prototyping of microfluidics was realised by high-precision milling of polycarbonate sheets, which offers flexibility and rapid turnover of the desired designs. On the other hand, the electrochemical sensor arrays were fabricated using standard photolithographic and metal (gold and silver) deposition technology in order to realise three-electrode cells comprising gold counter and working electrodes as well as silver reference electrode. The integration of fluidic chips and electrode arrays was realised via a laser-machined double-sided adhesive gasket that allowed creating the microchannels necessary for sample and reagent delivery. We focused our attention on the reproducibility of the electrode array preparation for the multiplexed detection of tumour markers such as carcinoembryonic antigen and prostate-specific antigen as well as genetic breast cancer markers such as estrogen receptor-alpha, plasminogen activator urokinase receptor, epidermal growth factor receptor and erythroblastic leukemia viral oncogene homolog 2. We showed that by carefully controlling the electrode surface pre-treatment and derivatisation via thiolated antibodies or short DNA probes that the detection of several key health parameters on a single chip was achievable with excellent reproducibility and high sensitivity.

Nosocomial transmission of hepatitis C virus during contrast-enhanced computed tomography scanning.

We have investigated two cases of acute hepatitis C that occurred in patients who underwent digestive endoscopy and contrast-enhanced computed tomography (CT) scanning at two different centers. Investigations to identify the sources of infection included an on-site review of diagnostic procedures, interview of the involved healthcare staff, serological testing of the patients who underwent the procedures before and after the index cases and a molecular analysis of viral isolates from the patients and from potential viremic sources. In both cases, the epidemiological investigation identified a chronic hepatitis C virus (HCV) carrier who had been subjected to CT-scanning immediately before the index patient. Genetic distance and molecular phylogenetic analyzes of HCV sequences showed a close relationship between the isolates from these carriers and those from the acute-hepatitis patients, strongly suggesting that patient-to-patient transmission had occurred during CT. This is the first report describing two well documented cases of HCV nosocomial patient-to-patient transmission during contrast-enhanced CT scanning.

The value of comparative research in major day surgery.

OBJECTIVE: To measure time trends in major day surgery rates according to hospital ownership and other hospital characteristics among the providers of the public healthcare network of Catalonia, Spain. METHOD: Data from the Statistics of Health Establishments providing Inpatient Care. A generalized linear mixed model with Gaussian response and random intercept and random slopes. RESULTS: The greatest growth in the rate of major day surgery was observed among private for-profit hospitals: 42.9 (SD: 22.5) in 2009 versus 2.7 (SD: 6.7) in 1996. These hospitals exhibited a significant increase in major day surgery compared to public hospitals (coefficient 2; p-value <0.01) CONCLUSIONS: The comparative evaluation of hospital performance is a decisive tool to ensure that public resources are used as rationally and efficiently as possible.

DNA biosensors based on gold nanoparticles-modified graphene oxide for the detection of breast cancer biomarkers for early diagnosis.

Two different DNA (ERBB2c and CD24c) modified gold nanoparticles and graphene oxide loaded on glassy carbon electrodes were prepared for early detection of breast cancer markers by electrochemical detection of HER2. Comparative study of ERBB2c and CD24c for the detection was carried out. A "sandwich-type" detection strategy was employed in this electrochemical DNA biosensor and its response was measured by amperometric detection. The electrochemical signal enhancement achieved via gold nanoparticles and grapheme oxide system allowed for sensitive detection of the breast cancer biomarker ERBB2 and the control marker CD24. The modified graphene oxide was characterised using Raman spectroscopy, UV-visible spectroscopy, Fourier transform infrared spectroscopy transmission electron microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The various steps involved in the modification of a glassy carbon electrode with graphene oxide, gold nanoparticles and DNA probes, target and reporter probe were electrochemically characterised using cyclic voltammetry and electrochemical impedance spectroscopy. Using amperometric detection of a horse radish peroxidase label, detection limits of 0.16nM and 0.23nM were obtained with sensitivity 378nA/nM and 219nA/nM for ERBB2 andCD24 respectively.

Feasibility and results of autologous stem cell transplantation in de novo acute myeloid leukemia in patients over 60 years old. Results of the CETLAM AML-99 protocol.

BACKGROUND AND OBJECTIVES: The benefits of high-dose cytarabine, anthracyclines and hematopoietic stem cell transplantation in the treatment of acute myeloid leukemia (AML) are greater in younger rather than in older patients. We assessed the proportion of patients over 60 years with de novo AML who qualified for intensive therapy and determined the feasibility and results of autologous stem cell transplantation (ASCT) in first complete remission (CR). DESIGN AND METHODS: Induction therapy included idarubicin, cytarabine and etoposide. Patients who achieved CR received one cycle of mitoxantrone and cytarabine and ASCT as consolidation therapies. RESULTS: Over a 4-year period, 258 patients were registered of whom 135 (52%) were enrolled for intensive treatment. The CR rate was 61%, advanced age (p=0.033) and unfavorable cytogenetics (p=0.015) emerged as independent negative prognostic factors for CR. The 2-year overall survival (OS) was 23 % (CI 14%-30%) and was poorer in patients with unfavorable cytogenetics (p=0.035), age over 70 years (p=0.019) or leukocytosis (p=0.006). Only 27% of the potential candidates underwent ASCT. The probability of 2-year leukemia-free survival after consolidation was 39% (CI 6%-71%) for these patients and 22% (CI 6% - 39%) for candidate patients not undergoing ASCT (p=0.07). INTERPRETATION AND CONCLUSIONS: Over 25% of the patients 60 to 70 years with de novo AML benefit from standard intensive treatment. In these patients, ASCT has a tolerable toxicity and may have a positive impact on leukemia-free survival.

Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 µL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 µL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers.