[Role of nurses specializing in oncology to support the care journey for patients with penile cancer]. / Rôle des infirmières coordinatrices en oncologie pour accompagner le trajet de soins des patients atteints d'un cancer de la verge.

The role of the specialized nurse in the management of penile cancer is essential to ensure quality care and appropriate support throughout the care pathway. Prior knowledge of the pathology seems essential to us. Organization, communication and education are essential to supporting patients. LEVEL OF EVIDENCE: 3.

Genome-wide meta-analysis of variant-by-diuretic interactions as modulators of lipid traits in persons of European and African ancestry.

Hypertension (HTN) is a significant risk factor for cardiovascular morbidity and mortality. Metabolic abnormalities, including adverse cholesterol and triglycerides (TG) profiles, are frequent comorbid findings with HTN and contribute to cardiovascular disease. Diuretics, which are used to treat HTN and heart failure, have been associated with worsening of fasting lipid concentrations. Genome-wide meta-analyses with 39,710 European-ancestry (EA) individuals and 9925 African-ancestry (AA) individuals were performed to identify genetic variants that modify the effect of loop or thiazide diuretic use on blood lipid concentrations. Both longitudinal and cross sectional data were used to compute cohort-specific interaction results, which were then combined through meta-analysis in each ancestry. These ancestry-specific results were further combined through trans-ancestry meta-analysis. Analysis of EA data identified two genome-wide significant (p < 5 × 10-8) loci with single nucleotide variant (SNV)-loop diuretic interaction on TG concentrations (including COL11A1). Analysis of AA data identified one genome-wide significant locus adjacent to BMP2 with SNV-loop diuretic interaction on TG concentrations. Trans-ancestry analysis strengthened evidence of association for SNV-loop diuretic interaction at two loci (KIAA1217 and BAALC). There were few significant SNV-thiazide diuretic interaction associations on TG concentrations and for either diuretic on cholesterol concentrations. Several promising loci were identified that may implicate biologic pathways that contribute to adverse metabolic side effects from diuretic therapy.

Variants in Pharmacokinetic Transporters and Glycemic Response to Metformin: A Metgen Meta-Analysis.

Therapeutic response to metformin, a first-line drug for type 2 diabetes (T2D), is highly variable, in part likely due to genetic factors. To date, metformin pharmacogenetic studies have mainly focused on the impact of variants in metformin transporter genes, with inconsistent results. To clarify the significance of these variants in glycemic response to metformin in T2D, we performed a large-scale meta-analysis across the cohorts of the Metformin Genetics Consortium (MetGen). Nine candidate polymorphisms in five transporter genes (organic cation transporter [OCT]1, OCT2, multidrug and toxin extrusion transporter [MATE]1, MATE2-K, and OCTN1) were analyzed in up to 7,968 individuals. None of the variants showed a significant effect on metformin response in the primary analysis, or in the exploratory secondary analyses, when patients were stratified according to possible confounding genotypes or prescribed a daily dose of metformin. Our results suggest that candidate transporter gene variants have little contribution to variability in glycemic response to metformin in T2D.

Case report: on the use of the HID-Ion AmpliSeq™ Ancestry Panel in a real forensic case.

In the absence of any other conclusive forensic evidence, DNA profiling is the method of choice for body identification. This study focuses on the case of a carbonized corpse whose complete autosomal short tandem repeat (STR) profile could not lead to direct identification by the investigators. To assist in the progress of investigation, we endeavoured to determine the biogeographical origin and eye colour of the deceased individual. Along with Y chromosome and mitochondrial DNA analyses, we applied a next-generation sequencing (NGS) approach to the study of ancestry informative markers (AIMs) using the HID-Ion AmpliSeq™ Ancestry Panel launched by Thermo Fisher Scientific. This work gave us the opportunity to test this new technology in a real forensic case. Although this study highlights the benefits of such a combined approach, as it markedly improves the specificity of the biogeographical profile, it also underlines the need for the accurate characterization of a larger collection of reference populations and the necessity of caution in data interpretation.

Targeted next generation sequencing application in cardiac channelopathies: Analysis of a cohort of autopsy-negative sudden unexplained deaths.

Genetic testing for cardiac channelopathies in sudden unexplained death (SUD) has developed substantially over the last years. The Next Generation Sequencing (NGS) technology provides an unprecedented opportunity to screen for genetic variations underlying arrhythmogenic genes in a short period of time at a low cost. The present study aimed to perform genetic testing with NGS technologies on the Ion Torrent Personal Genome Machine™ (Ion PGM™) sequencer, in targeting a total of 23 genes reported to be associated with inherited cardiac channelopathies in order to identify the possible cause of death in a cohort of post-mortem cases. The molecular analyses focused on 16 cases of SUD, aged less than 35 years old. In all cases, the cause of death could not be determined after a rigorous autopsy associated with histopathological and toxicological analyses according to the guidelines of the Association for European Cardiovascular Pathology. DNA was extracted from fresh frozen tissue. An average of 200 variants was identified per case. However, after the prioritization process using a new scoring program (VaRank) and after the conjunction of clinical data and molecular findings, four "likely pathogenic" variants (including two undescribed variants), were identified in three cases (18.75%) of our cohort in the genes KCNH2, ANK2, SCN5A and RYR2. One case, who died during psychiatric hospitalization after administration of a QT prolonging drug, showed a double "likely pathogenic" variant in Long QT genes (ANK2 and SCN5A) which may have predisposed to drug-induced cardiac arrhythmias. Our study illustrates that the NGS approach based on AmpliSeq™ libraries and Ion Torrent PGM™ sequencing may be an efficient approach, integrated to post-mortem examination. Given the massive amount of information generated by NGS, a rigorous filtration strategy of variants coupled with multidisciplinary collaboration is crucial to determine the potential pathogenic role of identified variants in the cause of death.

RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise.

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.

Novel contribution on the diagenetic physicochemical features of bone and teeth minerals, as substrates for ancient DNA typing.

The extraction of DNA from skeletal remains is a major step in archeological or forensic contexts. However, diagenesis of mineralized tissues often compromises this task although bones and teeth may represent preservation niches allowing DNA to persist over a wide timescale. This exceptional persistence is not only explained on the basis of complex organo-mineral interactions through DNA adsorption on apatite crystals composing the mineral part of bones and teeth but is also linked to environmental factors such as low temperatures and/or a dry environment. The preservation of the apatite phase itself, as an adsorption substrate, is another crucial factor susceptible to significantly impact the retrieval of DNA. With the view to bring physicochemical evidence of the preservation or alteration of diagenetic biominerals, we developed here an analytical approach on various skeletal specimens (ranging from ancient archeological samples to recent forensic specimens), allowing us to highlight several diagenetic indices so as to better apprehend the complexity of bone diagenesis. Based on complementary techniques (X-ray diffraction (XRD), Fourier transform infrared (FTIR), calcium and phosphate titrations, SEM-EDX, and gravimetry), we have identified specific indices that allow differentiating 11 biological samples, primarily according to the crystallinity and maturation state of the apatite phase. A good correlation was found between FTIR results from the analysis of the v3(PO4) and v4(PO4) vibrational domains and XRD-based crystallinity features. A maximal amount of information has been sought from this analytical approach, by way of optimized posttreatment of the data (spectral subtraction and enhancement of curve-fitting parameters). The good overall agreement found between all techniques leads to a rather complete picture of the diagenetic changes undergone by these 11 skeletal specimens. Although the heterogeneity and scarcity of the studied samples did not allow us to seek direct correlations with DNA persistence, the physicochemical parameters described in this work permit a fine differentiation of key properties of apatite crystals among post mortem samples. As a perspective, this analytical approach could be extended to more numerous sets of specimens so as to draw statistical relationships between mineral and molecular conservation.

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise.

The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based single nucleotide polymorphism genotyping assay using iPLEX gold technology for identification of Mycobacterium tuberculosis complex species and lineages.

The major goal of the present study was to investigate the potential use of a novel single nucleotide polymorphism (SNP) genotyping technology, called iPLEX Gold (Sequenom), for the simultaneous analysis of 16 SNPs that have been previously validated as useful for identification of Mycobacterium tuberculosis complex (MTBC) species and classification of MTBC isolates into distinct genetic lineages, known as principal genetic groups (PGGs) and SNP cluster groups (SCGs). In this context, we developed a 16-plex iPLEX assay based on an allele-specific-primer single-base-extension reaction using the iPLEX Gold kit (Sequenom), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis on the commercially available Sequenom MassARRAY platform. This assay was tested on a panel of 55 well-characterized MTBC strains that were also genotyped for the same loci using the previously reported SNaPshot assay, as well as 10 non-MTBC mycobacteria and 4 bacteria not belonging to the genus Mycobacterium. All MTBC samples were successfully analyzed with the iPLEX assay, which yielded clear allelic data for 99.9% of the SNPs (879 out of 880). No false-positive results were obtained with the negative controls. Compared to the SNaPshot assay, the newly developed 16-plex iPLEX assay produced fully concordant results that allowed reliable differentiation of MTBC species and recognition of lineages, thus demonstrating its potential value in diagnostic, epidemiological, and evolutionary applications. Compared to the SNaPshot approach, the implementation of the iPLEX technology could offer a higher throughput and could be a more flexible and cost-effective option for microbiology laboratories.

Single nucleotide polymorphisms in genes that are associated with a modified response to statin therapy: the Rotterdam Study.

The objective of this study was to investigate whether common variation in genes involved in lipid metabolism modify the effect of statins on serum total cholesterol concentration. Statin users were identified in the Rotterdam Study, a prospective population-based cohort study of subjects >55 years of age. We studied the association between single nucleotide polymorphisms (SNPs) in genes involved in lipid metabolism and total cholesterol response to statin therapy, using linear regression analysis and adjusting for potential confounders. Replication was performed in an independent extended cohort of the Rotterdam Study. Genotype data and total cholesterol concentrations after start of statin therapy were available for 554 newly started statin users. Two SNPs were associated with a significantly higher cholesterol concentration under statin therapy: SNP rs1532624 in the CETP gene (ß: 0.141 mmol l(-1), P=0.004 per additional allele) and SNP rs533556 in the APOA1 gene (ß: 0.138 mmol l(-1), P=0.005 per additional allele). In the replication sample, only the CETP rs1532624 SNP again showed a significant association. The SNPs were not related to baseline total cholesterol in non-statin users. In conclusion, we found that the CETP rs1532624 polymorphism is associated with cholesterol response to statin therapy in a cohort of elderly subjects in the general population.

Identification and genotyping of Mycobacterium tuberculosis complex species by use of a SNaPshot Minisequencing-based assay.

The aim of the present study was to investigate the use of the SNaPshot minisequencing method for the identification of Mycobacterium tuberculosis complex (MTBC) isolates to the species level and for further genotyping of M. tuberculosis isolates. We developed an innovative strategy based on two multiplex allele-specific minisequencing assays that allowed detection of eight species-specific and eight lineage-specific single nucleotide polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification, followed by an eightplex minisequencing reaction with the SNaPshot multiplex kit (Applied Biosystems) and, finally, analysis of the extension products by capillary electrophoresis. The whole strategy was developed with a panel of 56 MTBC strains and 15 negative controls. All MTBC strains tested except one M. africanum clinical isolate were accurately identified to the species level, and all M. tuberculosis isolates were successfully further genotyped. This two-step strategy based on SNaPshot minisequencing allows the simultaneous differentiation of closely related members of the MTBC, the distinction between principal genetic groups, and the characterization of M. tuberculosis isolates into one of the seven prominent SNP cluster groups (SCGs) and could be a useful tool for diagnostic and epidemiological purposes.

Characterization of Metarhizium species and varieties based on molecular analysis, heat tolerance and cold activity.

AIMS: The genetic relationships and conidial tolerances to high and low temperatures were determined for isolates of several Metarhizium species and varieties. METHODS AND RESULTS: Molecular-based techniques [AFLP and rDNA (ITS1, ITS2 and 5.8S) gene sequencing] were used to characterize morphologically identified Metarhizium spp. isolates from a wide range of sources. Conidial suspensions of isolates were exposed to wet heat (45 + or - 0.2 degrees C) and plated on potato dextrose agar plus yeast extract (PDAY) medium. After 8-h exposure, the isolates divided clearly into two groups: (i) all isolates of Metarhizium anisopliae var. anisopliae (Ma-an) and Metarhizium from the flavoviride complex (Mf) had virtually zero conidial relative germination (RG), (ii) Metarhizium anisopliae var. acridum (Ma-ac) isolates demonstrated high heat tolerance (c. 70-100% RG). Conidial suspensions also were plated on PDAY and incubated at 5 degrees C for 15 days, during which time RGs for Ma-an and Ma-ac isolates were virtually zero, whereas the two Mf were highly cold active (100% RG). CONCLUSIONS: Heat and cold exposures can be used as rapid tools to tentatively identify some important Metarhizium species and varieties. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of Metarhizium spp. currently relies primarily on DNA-based methods; we suggest a simple temperature-based screen to quickly obtain tentative identification of isolates as to species or species complexes.

Efficiency evaluation of a DNA extraction and purification protocol on archival formalin-fixed and paraffin-embedded tissue.

Formalin-fixed and paraffin-embedded tissue (FF-PET) is an invaluable resource for retrospective molecular genetic studies, but the extraction of high-quality genomic DNA from FF-PET is still a problematic issue. Despite the range of DNA extraction methods currently in use, the association of phenol-chloroform extraction and silica-based purification protocols, reported in ancient DNA studies on archaeological bones, has, to our knowledge, not been used for DNA extraction from FF-PET yet. The present study compared the efficiency of three DNA extraction and purification protocols from two different FF-PET substrates, heart and liver, by using quantitative PCR and multiplex amplification. We showed that the method, using phenol-chloroform and the QIAamp DNA mini Kit (Qiagen), was the most effective DNA extraction and purification method and that the DNA quantity extracted from liver is statistically more important than that extracted from heart. Autosomal STR typing by multiplex amplifications gave partial allelic profiles with only small size products (less than 300 bases) amplified, suggesting that DNA extracted from FF-PET was degraded. In conclusion, the protocol presented here, previously described in studies on ancient bones, should find application in different molecular studies involving FF-PET material.

[Thyroid cancers]. / Les cancers thyroïdiens.

During the last two decades, the incidence of thyroid cancer has doubled, mainly do to the early detection of small papillary tumors. However, mortality stayed stable (0.05/100.000). Well differentiated cancers (papillary and follicular) demonstrated a excellent survival prognosis (95 % at 30 years for the majority of the patients), factors of prognosis: age, size of the initial tumor, presence (or not) of distant metastases, lymph node involvement (only in patients 45 year or older). Surgery is the main treatment and should be with curative intent, hence the importance of a thorough preoperative work-up: sonography, needle aspiration cytology and MRI of cervicomediastinum for large tumors and/or suspected lymph nodes. Total thyroidectomy is mandatory, excepted for well selected patients with small (pT1) unilateral tumors under the age of 45. Central compartment lymph node clearance is advocated (but not evidence based) with presentation of the recurrent laryngeal nerves and of the parathyroids. Ablation of residual thyroid tissue (Iode 131) should be advocated for patients at high (or intermediary) risk of recurrence, only after multidisciplinary concertation. Unlimited follow-up checking the thyroglobulin serum local after the total thyroidectomy and radioactive ablation, the availability of recombinant rhTSH avoids the withdrawal of thyroid hormones (hypothyroid period). Anaplastic carcinomas are very aggressive, no therapeutic solution, excepted in some selected case for when radical surgery is possible (flap reconstruction) followed by chemoradiation. Calcitoninenia to detect and to follow medullary thyroid cancer after total thyroidectomy and lymph node clearance.

First successful assay of Y-SNP typing by SNaPshot minisequencing on ancient DNA.

In the present study, a set of 13 Y-chromosomal single nucleotide polymorphisms (Y-SNPs) selected for the identification of the most frequent Asian Y-haplogroups was included in an allele-specific primer extension assay. Single nucleotide polymorphism (SNP) genotyping was accomplished by co-amplification of these 13 DNA fragments within 2 multiplex PCRs followed by detection with 1 minisequencing reaction using the SNaPshottrade mark Multiplex kit and analysis of extension products by capillary electrophoresis. First developed on modern samples, the assay was optimized for the analysis of 11 ancient DNA (aDNA) samples from the Krasnoyarsk region (southern Siberia) that were dated from 5,500-1,800 years before present (YBP). SNP typing was successful for most of them, which were all assigned to Y-haplogroup R1a1 except one. These results show that SNPs are well-suited for the analysis of aged and degraded DNA samples. Moreover, we found that the SNaPshot minisequencing methodology is a convenient, robust, and efficient method for SNP typing. To our knowledge, this study reports the first successful investigation of Y-SNPs on aDNA samples. The potential use of Y-SNPs in both evolutionary and forensic fields is also discussed.

STR typing of ancient DNA extracted from hair shafts of Siberian mummies.

The aim of this study was to determine if ancient hair shafts could be suitable for nuclear DNA analysis and to develop an efficient and straightforward protocol for DNA extraction and STR typing of ancient specimens. The developed method was validated on modern and forensic samples and then successfully applied on ancient hairs collected from Siberian mummies dating from the 16th to the early 19th centuries. In parallel extractions including or excluding a washing step were performed at least two times for each sample in order to evaluate the influence on the quantity of nuclear DNA yielded and on the typing efficiency. Twelve ancient individuals were analyzed through our approach and full and reliable profiles were obtained for four of them. These profiles were validated by comparison with those obtained from bone and teeth DNA extracted from the same ancient specimens. The present study demonstrates that the washing step cannot be considered as deleterious for DNA retrieval since the same results were obtained by the two approaches. This finding challenges the hypothesis that recoverable nuclear DNA is only found on the outer surface of hair shafts and provides evidence that nuclear DNA can be successfully extracted from ancient hair shafts. The method described here constitutes a promising way for non-invasive investigations in ancient DNA analysis for precious or historical samples as well as forensic casework analyses.

Early influence of the steppe tribes in the peopling of Siberia.

The Yakuts, Middle Age Turkic speakers (15th-16th centuries), are widely accepted as the first settlers of the Altai-Baikal area in eastern Siberia. They are supposed to have introduced horses and developed metallurgy in this geographic area during the 15th or 16th century A.D. The analysis of the Siberian grave of Pokrovsk, recently discovered near the Lena River (61 degrees 29' N) and dated by accelerator mass spectrometry from 2,400 to 2,200 years B.P., may provide new elements to test this hypothesis. The exceptional combination of various artifacts and the mitochondrial DNA data extracted from the bone remains of the Pokrovsk man might prove the existence of previous contacts between autochthonous hunters of Oriental Siberia and the nomadic horse breeders from the Altai-Baikal area (Mongolia and Buryatia). Indeed, the stone arrowhead and the harpoons relate this Pokrovsk man to the traditional hunters of the Taiga. Some artifacts made of horse bone and the pieces of armor, however, are related to the tribes of Mongolia and Buryatia of the Xiongnu period (3rd century B.C.). This affinity has been confirmed by the match of the mitochondrial haplotype of this subject with a woman of the Egyin Gol necropolis (Mongolia, 2nd/3rd century A.D.) as well as with two modern Buryats. This result allows us to postulate that contacts between southern steppe populations and Siberian tribes occurred before the 15th century.

Genetic analysis of human remains from a double inhumation in a frozen kurgan in Kazakhstan (Berel site, Early 3rd Century BC).

The discovery of a big barrow of the Saka period in eastern Kazakhstan between the Russian and the Chinese borders provided the opportunity to excavate a frozen burial site. In the burial chamber, there was a wooden sarcophagus with two human bodies. The skeletons of these two individuals, a man and a woman, were well preserved. A genetic study based on STRs and mitochondrial DNA analyses was undertaken in order to determine whether these human remains belonged to close relatives. Results were obtained for all the markers. Nevertheless, nuclear STRs did not allow a clear conclusion concerning the relationship, but analysis of mitochondrial DNA showed that these skeletons were not close relatives.

Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur.

Although skeletal pain plays a major role in reducing the quality of life in patients suffering from osteoarthritis, Paget's disease, sickle cell anemia and bone cancer, little is known about the mechanisms that generate and maintain this pain. To define the peripheral fibers involved in transmitting and modulating skeletal pain, we used immunohistochemistry with antigen retrieval, confocal microscopy and three-dimensional image reconstruction of the bone to examine the sensory and sympathetic innervation of mineralized bone, bone marrow and periosteum of the normal mouse femur. Thinly myelinated and unmyelinated peptidergic sensory fibers were labeled with antibodies raised against calcitonin gene-related peptide (CGRP) and the unmyelinated, non-peptidergic sensory fibers were labeled with the isolectin B4 (Bandeira simplicifolia). Myelinated sensory fibers were labeled with an antibody raised against 200-kDa neurofilament H (clone RT-97). Sympathetic fibers were labeled with an antibody raised against tyrosine hydroxylase. CGRP, RT-97, and tyrosine hydroxylase immunoreactive fibers, but not isolectin B4 positive fibers, were present throughout the bone marrow, mineralized bone and the periosteum. While the periosteum is the most densely innervated tissue, when the total volume of each tissue is considered, the bone marrow receives the greatest total number of sensory and sympathetic fibers followed by mineralized bone and then periosteum. Understanding the sensory and sympathetic innervation of bone should provide a better understanding of the mechanisms that drive bone pain and aid in developing therapeutic strategies for treating skeletal pain.