High Molecular Variability Research Articles

Emerging Perspectives in Zinc Transporter Research in Prostate Cancer: An Updated Review.

Dysregulation of zinc and zinc transporters families has been associated with the genesis and progression of prostate cancer. The prostate epithelium utilizes two types of zinc transporters, the ZIP (Zrt-, Irt-related Protein) and the ZnTs (Zinc Transporter), to transport zinc from the blood plasma to the gland lumen. ZIP transporters uptake zinc from extracellular space and organelle lumen, while ZnT transporters release zinc outside the cells or to organelle lumen. In prostate cancer, a commonly observed low zinc concentration in prostate tissue has been correlated with downregulations of certain ZIPs (e.g., ZIP1, ZIP2, ZIP3, ZIP14) and upregulations of specific ZnTs (e.g., ZnT1, ZnT9, ZnT10). These alterations may enable cancer cells to adapt to toxic high zinc levels. While zinc supplementation has been suggested as a potential therapy for this type of cancer, studies have yielded inconsistent results because some trials have indicated that zinc supplementation could exacerbate cancer risk. The reason for this discrepancy remains unclear, but given the high molecular and genetic variability present in prostate tumors, it is plausible that some zinc transporters-comprising 14 ZIP and 10 ZnT members-could be dysregulated in others patterns that promote cancer. From this perspective, this review highlights novel dysregulation, such as ZIP-Up/ZnT-Down, observed in prostate cancer cell lines for ZIP4, ZIP8, ZnT2, ZnT4, ZnT5, etc. Additionally, an in silico analysis of an available microarray from mouse models of prostate cancer (Nkx3.1;Pten) predicts similar dysregulation pattern for ZIP4, ZIP8, and ZnT2, which appear in early stages of prostate cancer progression. Furthermore, similar dysregulation patterns are supported by an in silico analysis of RNA-seq data from human cancer tumors available in cBioPortal. We discuss how these dysregulations of zinc transporters could impact zinc supplementation trials, particularly focusing on how the ZIP-Up/ZnT-Down dysregulation through various mechanisms might promote prostate cancer progression.

Morphological and genetic diversity of maize landraces along an altitudinal gradient in the Southern Andes.

Maize (Zea mays ssp. mays) is a major cereal crop worldwide and is traditionally or commercially cultivated almost all over the Americas. The North-Western Argentina (NWA) region constitutes one of the main diversity hotspots of the Southern Andes, with contrasting landscapes and a large number of landraces. Despite the extensive collections performed by the "Banco Activo de Germoplasma INTA Pergamino, Argentina" (BAP), most of them have not been characterized yet. Here we report the morphological and molecular evaluation of 30 accessions collected from NWA, along an altitudinal gradient between 1120 and 2950 meters above sea level (masl). Assessment of morphological variation in a common garden allowed the discrimination of two groups, which differed mainly in endosperm type and overall plant size. Although the groups retrieved by the molecular analyses were not consistent with morphological clusters, they showed a clear pattern of altitudinal structuring. Affinities among accessions were not in accordance with racial assignments. Overall, our results revealed that there are two maize gene pools co-existing in NWA, probably resulting from various waves of maize introduction in pre-Columbian times as well as from the adoption of modern varieties by local farmers. In conclusion, the NWA maize landraces preserved at the BAP possess high morphological and molecular variability. Our results highlight their potential as a source of diversity for increasing the genetic basis of breeding programs and provide useful information to guide future sampling and conservation efforts.

Molecular variability of Macrophomina phaseolina isolates using SSR marker

An experiment was conducted at RARI, Durgapura, SKNAU, Jobner during kharif 2020 to analyze the genetic diversity of the 12 Macrophomina phaseolina isolates by using simple sequence repeat (SSR) markers. The SSR primers were tested for amplification of genomic DNA of Macrophomia phaseolina isolates. Total five primers of MB series were screened against twelve Macrophomia phaseolina isolates. All primes amplified scorable banding pattern. Level of polymorphism for SSR analysis ranged between 60–85.7%. The similarity matrix indicated that 12 isolates exhibited 42.82–100% similarity coefficient. The SSR analysis shows high molecular variability among the isolates grouped into five major clusters A, B, C, D and E. In the similarity matrix Mp-DPR was found to have higher value of similarity coefficient (1), whereas Mp-JSM was found to have lower value if similarity coefficient (0.42).

Morphological and molecular characterization of Croatian carob tree (Ceratonia siliqua L.) germplasm

The results of morphological and AFLP variability of 120 plants of carob tree (Ceratonia siliqua L.), collected from 12 different locations (10 biological replicates for each location) on the coast and islands of the southern Croatian Adriatic, indicate high molecular and morphological variability among these carob populations. Analysis of molecular variance revealed significant differences among populations (26.07%; p < 0.001; a = 0.05). Out of the total variability, 22.49% refers to the variability among, and 77.51% within populations. UPGMA and STRUCTURE analysis based on AFLP genetic data clustered carob populations into three main groups representing three real genetic populations. UPGMA analysis based on morphological traits of leaves, pods, and seeds clustered carob populations into five groups. Mantel test showed significant correlation between morphological and genetic data (r = 0.58, p < 0.001; a = 0.05). According to the high genetic and morphological variability, the germplasm collection in the analysis could represent an important genetic pool for future breeding programmes. The goal of future research should be the conservation of C. siliqua in its natural habitats, and the establishment of gene banks of genetic resources with the purpose of creating new carob cultivars in breeding programmes.

High molecular variability in three pine vole species of the subgenus Terricola ( Microtus , Arvicolinae) and plausible source of polymorphism

High molecular variability in three pine vole species of the subgenus <i>Terricola</i> ( <i>Microtus</i> , Arvicolinae) and plausible source of polymorphism

Infra-specific variation of Acer cappadocicum (Sapindaceae): morphological and molecular approaches

Besides Quercus, Acer L. genus is the largest tree genus in the Northern Hemisphere and consists of about 124–156 species. High morphological and molecular variability has led to infra-specific complexity within most species of this genus. In Iran, the Acer cappadocicum subsp. cappadocicum Gled. is distributed in eastern to western parts of the Hyrcanian forests. In this study, three leaf-morphotypes in A. cappadocicum Gled. were identified during sampling in a forest located in the Hyrcanian region, leading to obscuring taxonomic situations of this taxon. Against this backdrop, the present study was performed to clarify intraspecific situation of this valuable tree using both macro-/micromorphological and molecular approaches. Forty-five plant samples of A. cappadocicum were randomly selected within nine geographical regions of four provinces in Hyrcanian forests of Iran. Internal transcribed spacer (ITS) (for eight individuals) and inter-simple sequence repeat (ISSR) molecular markers (for 45 individuals) were used along with the epidermal scanning electron micrograph (SEM) of the leaf epidermis. Considering the obtained results, acceptance of the expected lower taxa in A. cappadocicum ssp. cappadocicum in north of Iran could not be supported by molecular evidence. Results of the study indicated no correlation between variations in leaf-morphology, ISSRs, and ITS. However, there are two main clusters in ITS tree without discrete morphological characters showing differentiation and variability in ITS region within this species. This subspecies was compared also with other subspecies.

Fraternine, a Novel Wasp Peptide, Protects against Motor Impairments in 6-OHDA Model of Parkinsonism.

Parkinson’s disease (PD) is a progressive neurodegenerative condition that affects the Central Nervous System (CNS). Insect venoms show high molecular variability and selectivity in the CNS of mammals and present potential for the development of new drugs for the treatment of PD. In this study, we isolated and identified a component of the venom of the social wasp Parachartergus fraternus and evaluated its neuroprotective activity in the murine model of PD. For this purpose, the venom was filtered and separated through HPLC; fractions were analyzed through mass spectrometry and the active fraction was identified as a novel peptide, called Fraternine. We performed two behavioral tests to evaluate motor discoordination, as well as an apomorphine-induced rotation test. We also conducted an immunohistochemical assay to assess protection in TH+ neurons in the Substantia Nigra (SN) region. Group treated with 10 μg/animal of Fraternine remained longer in the rotarod compared to the lesioned group. In the apomorphine test, Fraternine decreased the number of rotations between treatments. This dose also inhibited dopaminergic neuronal loss, as indicated by immunohistochemical analysis. This study identified a novel peptide able to prevent the death of dopaminergic neurons of the SN and recover motor deficit in a 6-OHDA-induced murine model of PD.

Exploring Alzheimer's Disease Molecular Variability via Calculation of Personalized Transcriptional Signatures.

Despite huge investments and major efforts to develop remedies for Alzheimer’s disease (AD) in the past decades, AD remains incurable. While evidence for molecular and phenotypic variability in AD have been accumulating, AD research still heavily relies on the search for AD-specific genetic/protein biomarkers that are expected to exhibit repetitive patterns throughout all patients. Thus, the classification of AD patients to different categories is expected to set the basis for the development of therapies that will be beneficial for subpopulations of patients. Here we explore the molecular heterogeneity among a large cohort of AD and non-demented brain samples, aiming to address the question whether AD-specific molecular biomarkers can progress our understanding of the disease and advance the development of anti-AD therapeutics. We studied 951 brain samples, obtained from up to 17 brain regions of 85 AD patients and 22 non-demented subjects. Utilizing an information-theoretic approach, we deciphered the brain sample-specific structures of altered transcriptional networks. Our in-depth analysis revealed that 7 subnetworks were repetitive in the 737 diseased and 214 non-demented brain samples. Each sample was characterized by a subset consisting of ~1–3 subnetworks out of 7, generating 52 distinct altered transcriptional signatures that characterized the 951 samples. We show that 30 different altered transcriptional signatures characterized solely AD samples and were not found in any of the non-demented samples. In contrast, the rest of the signatures characterized different subsets of sample types, demonstrating the high molecular variability and complexity of gene expression in AD. Importantly, different AD patients exhibiting similar expression levels of AD biomarkers harbored distinct altered transcriptional networks. Our results emphasize the need to expand the biomarker-based stratification to patient-specific transcriptional signature identification for improved AD diagnosis and for the development of subclass-specific future treatment.

Proteins involved in the biosynthesis of lipophosphoglycan in Leishmania: a comparative genomic and evolutionary analysis

BackgroundLeishmania spp. are digenetic parasites capable of infecting humans and causing a range of diseases collectively known as leishmaniasis. The main mechanisms involved in the development and permanence of this pathology are linked to evasion of the immune response. Crosstalk between the immune system and particularities of each pathogenic species is associated with diverse disease manifestations. Lipophosphoglycan (LPG), one of the most important molecules present on the surface of Leishmania parasites, is divided into four regions with high molecular variability. Although LPG plays an important role in host-pathogen and vector-parasite interactions, the distribution and phylogenetic relatedness of the genes responsible for its synthesis remain poorly explored. The recent availability of full genomes and transcriptomes of Leishmania parasites offers an opportunity to leverage insight on how LPG-related genes are distributed and expressed by these pathogens.ResultsUsing a phylogenomics-based framework, we identified a catalog of genes involved in LPG biosynthesis across 22 species of Leishmania from the subgenera Viannia and Leishmania, as well as 5 non-Leishmania trypanosomatids. The evolutionary relationships of these genes across species were also evaluated. Nine genes related to the production of the glycosylphosphatidylinositol (GPI)-anchor were highly conserved among compared species, whereas 22 genes related to the synthesis of the repeat unit presented variable conservation. Extensive gain/loss events were verified, particularly in genes SCG1-4 and SCA1-2. These genes act, respectively, on the synthesis of the side chain attached to phosphoglycans and in the transfer of arabinose residues. Phylogenetic analyses disclosed evolutionary patterns reflective of differences in host specialization, geographic origin and disease manifestation.ConclusionsThe multiple gene gain/loss events identified by genomic data mining help to explain some of the observed intra- and interspecies variation in LPG structure. Collectively, our results provide a comprehensive catalog that details how LPG-related genes evolved in the Leishmania parasite specialization process.

Multiple haplotypes of Echinococcus granulosus sensu stricto in single naturally infected intermediate hosts.

Cystic echinococcosis is a disease that affects both humans and animals, caused by cryptic species complex belonging to the platyhelminth Echinococcus granulosus sensu lato (s.l.). This disease is distributed worldwide, with E. granulosus sensu stricto (s.s.) being the most widespread of the species. High genetic variability has been demonstrated within E. granulosus s.s. studying single cyst per infected animal identifying a number of different haplotypes. However, few studies have addressed the genetic diversity of this parasite within a single intermediate host with multiple Echinococcus cysts. To date, it remains unknown if specific haplotypes of E. granulosus s.s. produce differences in biological features of the cyst. Here, we use the full length of the mitochondrial gene cox1 to determine E. granulosus s.s. haplotypes in samples from both cattle and sheep which harboured more than one cyst in different areas in Chile, where this parasite is endemic. We found 16 different haplotypes in 66 echinococcal cysts from 10 animals, and both cattle and sheep can harbour up to five different haplotypes of E. granulosus s.s. in the same animal. Regarding cyst fertility, five animals had both fertile and infertile Echinococcus cysts in both single and multiple haplotype infections. There was no association between haplotype and cyst fertility, size, or adventitial layer characteristics. Sampling and sequencing every Echinococcus cyst found in the intermediate host reveals a high molecular variability. We speculate that multiple haplotype infections could also suggest that intermediate hosts come from hyperendemic areas.

Chromosome variability and evolution in rodents of the tribe Abrotrichini (Rodentia, Cricetidae, Sigmodontinae)

Rodents are a very diverse group with large chromosome variability. One of the most species rich linage in the Neotropics is the Sigmodontinae. Among them, the tribe Abrotrichini was recently defined and its taxonomy and phylogeny were mostly elucidated through molecular and morphological evidence. Meanwhile, chromosome data were only secondarily used because of fragmentary information. In this contribution, we conduct a chromosome characterization of Abrothrix hirta, A. olivacea, A. andina, and Paynomys macronyx, review the cytogenetic background of the tribe, and contrast it with molecular data. Chromosomes were analyzed by conventional and differential techniques. All Abrothrix species presented 2n = 52/FNa = 56, with a high similarity in the banding patterns reflecting a conserved karyotype, which does not coincide with its high molecular variability. In turn, P. macronyx have 2n = 54/FNa = 58–59, varying due to a heteromorphic pair of autosomes. In addition, in this last species, different morphologies of the X chromosome and the presence of B chromosomes were detected. Heterochromatin was involved in these variants. The telomeric probe in P. macronyx marks terminal regions of all chromosomes. B chromosomes generated strong telomeric signals. The Ag-NORs banding revealed the same patterns in Abrothrix and Paynomys. Cytogenetic data support phylogenetic relationships previously proposed and suggest that the specious genus Abrothrix could have retained the ancestral karyotype of the subfamily. In the tribe, the relatively conserved chromosome complement contrasts with its high molecular variability. This indicates decoupling between the rates of chromosomal and molecular divergence, as observed in other rodent lineages. In abrotrichines, chromosome evolution was slower.

Evidence for long‐term prevalence of cucumber vein yellowing virus in Sudan and genetic variation of the virus in Sudan and the Mediterranean Basin

Cucumber vein yellowing virus (CVYV) is emerging throughout the Mediterranean Basin, where it causes significant damage to cucumber and melon crops. It has been suggested that CVYV originated from the Middle East and has spread only recently to other areas. In this work, an isolate from Sudan was characterized, and surveys performed in that country between 1992 and 2012 revealed a long‐term presence of CVYV with a high molecular variability, showing that the virus has long been endemic in sub‐Saharan Africa. Comparison of the full‐length sequences of 11 CVYV isolates from different geographic origins revealed recombination events in CVYV populations from the Mediterranean Basin and the Middle East, and evidence for different selection pressures along the genome. These results shed a new light on the evolution of CVYV.

Molecular and Aggressiveness Characterization of Isolates of Fusarium solani and Fusarium oxysporum f.sp. passiflorae Associated to Passion Fruit Wilting

This work was carried out with the objective of performing a molecular and aggressiveness characterization of F. solani and F. oxysporum f.sp. passiflorae collected in the Pantanal, Cerrado and Amazon biomes. We selected the most aggressive isolates for use in breeding programs aiming resistance to Collar Rot and Fusariosis. For inoculation of the isolates of F. oxysporum f.sp. passiflorae the washed root method was used. The molecular characterization of the isolates was carried out by partial sequencing of the Transcribed Internal Spacer of the rDNA region. The isolates of F. solani formed two distinct groups in relation to aggressiveness. Among all isolates, FSUNEMAT 40 and FSUNEMAT 46 were the most aggressive. The model with K=2 was taken as the best model to explain the genetic structure of the F. solani populations, with clear combinations of genes from both gene pools. There were three groups with respect to the aggressiveness of the isolates of F. oxysporum f.sp. passiflorae, with the isolated FOUNEMAT 22 being the most aggressive. In view of these results, the isolates of F. solani collected in P. edulis in the state of Mato Grosso presented a high molecular variability independent from the biome of origin, and this was also observed in the tests of aggressiveness. The results indicate the need to consider the molecular variability and the aggressiveness of the pathogens in the evaluation of genotypes of sour passion fruit in programs of selection of resistant cultivars.

Cellular network modeling and single cell gene expression analysis reveals novel hepatic stellate cell phenotypes controlling liver regeneration dynamics

BackgroundRecent results from single cell gene and protein regulation studies are starting to uncover the previously underappreciated fact that individual cells within a population exhibit high variability in the expression of mRNA and proteins (i.e., molecular variability). By combining cellular network modeling, and high-throughput gene expression measurements in single cells, we seek to reconcile the high molecular variability in single cells with the relatively low variability in tissue-scale gene and protein expression and the highly coordinated functional responses of tissues to physiological challenges. In this study, we focus on relating the dynamic changes in distributions of hepatic stellate cell (HSC) functional phenotypes to the tightly regulated physiological response of liver regeneration.ResultsWe develop a mathematical model describing contributions of HSC functional phenotype populations to liver regeneration and test model predictions through isolation and transcriptional characterization of single HSCs. We identify and characterize four HSC transcriptional states contributing to liver regeneration, two of which are described for the first time in this work. We show that HSC state populations change in vivo in response to acute challenges (in this case, 70% partial hepatectomy) and chronic challenges (chronic ethanol consumption). Our results indicate that HSCs influence the dynamics of liver regeneration through steady-state tissue preconditioning prior to an acute insult and through dynamic control of cell state balances. Furthermore, our modeling approach provides a framework to understand how balances among cell states influence tissue dynamics.ConclusionsTaken together, our combined modeling and experimental studies reveal novel HSC transcriptional states and indicate that baseline differences in HSC phenotypes as well as a dynamic balance of transitions between these phenotypes control liver regeneration responses.

Root Infection of Canker Pathogens, Fusarium circinatum and Diplodia sapinea, in Asymptomatic Trees in Pinus radiata and Pinus pinaster Plantations

The existence of a latent stage within host tissue of the pine pathogens Fusarium circinatum and Diplodia sapinea, the causal agents of pitch canker and shoot blight disease respectively, has previously been cited. However, studies on this cryptic phase in each disease lifecycle has only been focused on the host aerial parts but not on the roots. Therefore, our objective was to analyze the presence of both pathogens in roots of non-symptomatic mature trees in plantations where the pathogens are known to be causing canker symptoms. For that, we sampled roots from ten non-symptomatic and ten symptomatic trees in three Pinus radiata and one Pinus pinaster plantations in Basque Country, Spain. Both pathogens were isolated from roots of non-symptomatic trees in a higher frequency than from roots of symptomatic trees, 23.3% and 6.6% respectively for D. sapinea and 16.6% and 3.3% respectively for F. circinatum. Neither pathogens was detected in the P. pinaster plantation. The two pathogens were never isolated from the same tree. A high molecular variability was observed for D. sapinea isolates with six different haplotypes and two mating types for the eleven characterized isolates, but only one haplotype and mating type was found for F. circinatum, with all isolates of both fungi being proved pathogenic. These results evidence the importance root infection may have in the disease lifecycle and, therefore, disease management.

Caracterización morfológica y molecular de poblaciones de pasto banderi

El pasto banderita [Bouteloua curtipendula (Michx.) Torr.] es una especie nativa de importancia ganadera; sin embargo, prácticas inadecuadas de pastoreo han reducido su diversidad genética. Se exploró y analizó la diversidad morfológica y genética de 51 poblaciones de pasto banderita del estado de Chihuahua. Las plantas se trasplantaron bajo condiciones de temporal. Dos años después se realizó la caracterización morfológica. Para evaluar la variabilidad genética se utilizaron marcadores moleculares AFLP. El análisis de componentes principales (ACP) mostró que los tres primeros componentes (CP), explican el 73.83 % de la variación. El CP1 presentó una correlación significativa (P&lt;0.0001) con rendimiento de materia seca (88 %), densidad de tallos (85 %), diámetro de macollo (83 %), altura de follaje (82 %), altura de planta (79 %) y longitud de hoja (65 %). El análisis de AFLP con cuatro pares de iniciadores detectó 186 bandas; 80.67 % (150 bandas) presentó polimorfismo. El mayor porcentaje de polimorfismo (93.75 %) y bandas polimórficas (48) se obtuvo con la combinación de iniciadores EcoRI-ACT+MseI-CTG. Las poblaciones de banderita presentaron alta variabilidad morfológica y molecular. Se detectaron ecotipos (444, 359 y 557) con potencial forrajero de acuerdo a su variabilidad morfológica y molecular para ser incluidos en programas de restauración de pastizales

&lt;b&gt;Characterization of race 65 of &lt;i&gt;Colletotrichum lindemuthianum&lt;/i&gt; by sequencing ITS regions

The present work aimed characterize isolates of C. lindemuthianum race 65 from different regions in Brazil by ITS sequencing. A total of 17 isolates of race 65, collected in the states of Mato Grosso, Minas Gerais, Parana, Santa Catarina and Sao Paulo, were studied. Analysis of the sequences of isolates 8, 9, 12, 14 and 15 revealed the presence of two single nucleotide polymorphisms (SNPs) in the ITS1 region at the same positions. These isolates, when analyzed together with the sequence of isolate 17, revealed a SNP in the ITS2 region. The highest genetic dissimilarity, observed between isolates 11 and 3 and between isolates 11 and 10, was 0.772. In turn, isolates 7 and 2 were the most similar, with a value of 0.002 for genetic distance. The phylogenetic tree obtained based on the sequences of the ITS1 and ITS2 regions revealed the formation of two groups, one with a subgroup. The results reveal high molecular variability among isolates of race 65 of C. lindemuthianum .

Angolan vegetable crops have unique genotypes of potential value for future breeding programmes

A survey was carried out in Angola with the aim of collecting vegetable crops. Collecting expeditions were conducted in Kwanza-Sul, Benguela, Huíla and Namibe Provinces and a total of 80 accessions belonging to 22 species was collected from farmers and local markets. Species belonging to the Solanaceae (37 accessions) and Cucurbitaceae (36 accessions) families were the most frequently found with pepper and eggplant being the predominant solanaceous crops collected. Peppers were sold in local markets as a mixture of different types, even different species: Capsicum chinense, C. baccatum, C. frutescens and C. pubescens. Most of the eggplant accessions collected belonged to Solanum aethiopicum L. Gilo Group, the so-called ‘scarlet eggplant’. Cucurbita genus was better represented than the other cucurbit crops. A high morphological variation was present in the Cucurbita maxima and C. moschata accessions. A set of 22 Cucurbita accessions from Angola, along with 32 Cucurbita controls from a wide range of origins, was cultivated in Valencia, Spain and characterised based on morphology and molecularity using a set of 15 microsatellite markers. A strong dependence on latitude was found in most of the accessions and as a result, many accessions did not set fruit. The molecular analysis showed high molecular variability and uniqueness in the collected accessions, as shown by their segregation from the set of global controls. In summary, the material collected is quite valuable because of its uniqueness and the potential of the breeding characteristics it possesses.

Agro-morphological and molecular diversity in castor (Ricinus communis L.) germplasm collected from Andaman and Nicobar Islands, India

Castor (Ricinus communis L.) is an industrial oilseed crop grown worldwide. Its oil with more than 80% ricinoleic acid makes it a chief raw material for numerous industrial applications and biofuel production. Castor grows in a wild form across India including Andaman & Nicobar Islands, which are geographically quite isolated from mainland India. Thirty-three accessions growing in isolation in these Islands were used in the present study. Genetic diversity among these accessions was assessed using 18 agro-morphological traits and 29 EST-SSR markers. High agro-morphological and molecular variability was observed among these accessions. Both agro-morphological traits and EST-SSR markers effectively discriminated the accessions. However, EST-SSRs separated the accessions into more groups than did agro-morphological data, implying high efficiency and resolution of EST-SSR markers in genetic analysis of castor germplasm from Andaman & Nicobar. The diverse accessions identified in the present investigation would serve as genetically diverse sources in castor breeding programmes.

ChemInform Abstract: Microbial Polysaccharide‐Based Membranes: Current and Future Applications

AbstractReview: 119 refs.