Bioactivity-Guided Screening of Wound-Healing Active Constituents from American Cockroach (Periplaneta americana).

Ethanol extract (EE) from Periplaneta americana (PA) is the main ingredient of Kangfuxin, which is a popular traditional chinese medicine (TCM) and has long been used for the clinical treatment of burns, wounds and ulcers. We compared the wound-healing activities of three extracts of PA using cutaneous wound-healing in mice as the bioactivity model. These three extracts were EE, total polysaccharide and total protein. We also tracked bioactive fractions in the EE by organic reagent extraction, column chromatography and HPLC. Seven compounds were successfully identified from the water elution fraction of the EE of PA using UPLC-MS. Among these compounds, four compounds (P2, P3, P4, P5(1)) were first reported in PA. Some of these compounds have been previously reported to have various pharmacological activities that could contribute to the high wound-healing activity of PA.

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system.

BACKGROUND: The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda. RESULTS: By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What's more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system. CONCLUSION: The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Dynamic change in spring habitat use by the brown eared pheasant (Crossptilon mantchuricum) in the Huanglong mountains, Yanan City, Shaanxi Province, China.

Characterization of foraging-site preferences of threatened and endangered species is a key component of effective habitat conservation. We studied foraging-site selection by the brown eared pheasant (Crossoptilon mantchuricum) in the Huanglongshan Nature Reserve, Yanan City, Shaanxi Province, China, from early February to end of May 2011. We identified feeding sites by locating tracks and scratches characteristic of the birds, and compared habitat characteristics at these sites to those at randomly selected sites across the study area. During the pre-breeding season, the birds tended to be found in the areas characterized by gullies within mixed forests with intermediate sun exposure on gentle slopes (< 10°), and close to water and footpaths. The sites utilized by the birds also featured greater tree diameter, lower shrub density, lower grass cover, and lower altitude than random sites. During the breeding season, the birds tended to be found in the areas of slightly higher altitude, more shrubs, moderately steep slopes (10°-20°), and farther from water and paths. These patterns were consistent with seasonal changes in vegetation and food-resource availability in the study area. Management of brown eared pheasants' populations for conservation must account for these seasonal shifts in habitat requirements.

Taxonomic position of Eothenomys wardi (Arvicolinae: Cricetidae) based on morphological and molecular analyses with a detailed description of the species.

Ward's Red-backed Vole (Eothenomys wardi) is a rodent from the family Cricetidae. This endemic species occurs only in extreme northwestern Yunnan province, China in the Mekong and Salween river divide. It occupies steep cliffs at 2,800 to 4,250 m above sea level on the remote Qinghai-Tibetan plateau. The validity of E. wardi is controversial and no specimens exist apart from the nominal series. In 2010, we collected 38 topotypes of E. wardi from Meri Snow Mountain. The results of our phylogenetic analyses based on nucleotide sequences of the mitochondrial genes cytochrome b (cytb) and cytochrome c oxidase subunit one (COI) suggest that E. wardi is the sister group of E. custos, against its previously presumed sister species or conspecific species E. chinensis. In addition, seven out of 34 morphological characters differentiate E. wardi from other members of the genus Eothenomys. Therefore, we consider E. wardi to be a valid species and we provide its detailed morphological description.

GenScalpel: an application for sequence retrieval and extraction from the GenBank flatfile.

GenScalpel is a program designed for the retrieval and extraction of specified sequences from large-scale sequence sets in NCBI GenBank flatfile format. This routine task in bioinformatics analysis is a pressing need for laboratory biologists. Another objective of application development is to respond to the new form of the NCBI Nucleotide Sequence Database, which was updated in November 2011. In addition to a powerful sequence refinement application, GenScalpel provides convenient functions for web-based sequence downloading or multiple files batch processing. This note discusses major applications of the program and includes example data sets to demonstrate its performance. The program is written in PERL. GenScalpel, including installation packages for Windows and Linux systems as well as the accompanying documentation, are available free of charge at http://genscalpel.biosv.com/.

Temperature and Diet Acclimation Modify the Acute Thermal Performance of the Largest Extant Amphibian.

The Chinese giant salamander (Andrias davidianus), one of the largest extant amphibian species, has dramatically declined in the wild. As an ectotherm, it may be further threatened by climate change. Therefore, understanding the thermal physiology of this species should be the priority to formulate related conservation strategies. In this study, the plasticity in metabolic rate and thermal tolerance limits of A. davidianus larvae were studied. Specifically, the larvae were acclimated to three temperature levels (7 °C, cold stress; 15 °C, optimum; and 25 °C, heat stress) and two diet items (red worm or fish fray) for 20 days. Our results indicated that cold-acclimated larvae showed increased metabolic capacity, while warm-acclimated larvae showed a decrease in metabolic capacity. These results suggested the existence of thermal compensation. Moreover, the thermal tolerance windows of cold-acclimated and warm-acclimated larvae shifted to cooler and hotter ranges, respectively. Metabolic capacity is not affected by diet but fish-fed larvae showed superiority in both cold and heat tolerance, potentially due to the input of greater nutrient loads. Overall, our results suggested a plastic thermal tolerance of A. davidianus in response to temperature and diet variations. These results are meaningful in guiding the conservation of this species.

Genomics and morphometrics reveal the adaptive evolution of pikas.

Pikas (Lagomorpha: Ochotonidae) are small mouse-like lagomorphs. To investigate their adaptation to different ecological environments during their dispersal from the Qinghai-Xizang (Tibet) Plateau (QTP), we collected 226 pikas and measured 20 morphological characteristics and recorded habitat information. We also sequenced the genome of 81 specimens, representing 27 putative pika species. The genome-wide tree based on 4 090 coding genes identified five subgenera, i.e., Alienauroa, Conothoa, Lagotona, Ochotona, and Pika, consistent with morphometric data. Morphologically, Alienauroa and Ochotona had similar traits, including smaller size and earlier divergence time compared to other pikas. Consistently, the habitats of Alienauroa and Ochotona differed from those of the remaining subgenera. Phylogenetic signal analysis detected 83 genes significantly related to morphological characteristics, including several visual and hearing-related genes. Analysis of shared amino acid substitutions and positively selected genes (PSGs) in Alienauroa and Ochotona identified two genes, i.e., mitochondrial function-related TSFM (p.Q155E) and low-light visual sensitivity-related PROM1 (p.H419Y). Functional experiments demonstrated that TSFM-155E significantly enhanced mitochondrial function compared to TSFM-155Q in other pikas, and PROM1-419Y decreased the modeling of dynamic intracellular chloride efflux upon calcium uptake. Alienauroa and Ochotona individuals mostly inhabit different environments (e.g., subtropical forests) than other pikas, suggesting that a shift from the larger ancestral type and changes in sensory acuity and energy enhancement may have been required in their new environments. This study increases our understanding of the evolutionary history of pikas.

A mitogenomic perspective on the ancient, rapid radiation in the Galliformes with an emphasis on the Phasianidae.

BACKGROUND: The Galliformes is a well-known and widely distributed Order in Aves. The phylogenetic relationships of galliform birds, especially the turkeys, grouse, chickens, quails, and pheasants, have been studied intensively, likely because of their close association with humans. Despite extensive studies, convergent morphological evolution and rapid radiation have resulted in conflicting hypotheses of phylogenetic relationships. Many internal nodes have remained ambiguous. RESULTS: We analyzed the complete mitochondrial (mt) genomes from 34 galliform species, including 14 new mt genomes and 20 published mt genomes, and obtained a single, robust tree. Most of the internal branches were relatively short and the terminal branches long suggesting an ancient, rapid radiation. The Megapodiidae formed the sister group to all other galliforms, followed in sequence by the Cracidae, Odontophoridae and Numididae. The remaining clade included the Phasianidae, Tetraonidae and Meleagrididae. The genus Arborophila was the sister group of the remaining taxa followed by Polyplectron. This was followed by two major clades: ((((Gallus, Bambusicola) Francolinus) (Coturnix, Alectoris)) Pavo) and (((((((Chrysolophus, Phasianus) Lophura) Syrmaticus) Perdix) Pucrasia) (Meleagris, Bonasa)) ((Lophophorus, Tetraophasis) Tragopan))). CONCLUSIONS: The traditional hypothesis of monophyletic lineages of pheasants, partridges, peafowls and tragopans was not supported in this study. Mitogenomic analyses recovered robust phylogenetic relationships and suggested that the Galliformes formed a model group for the study of morphological and behavioral evolution.

The complete mitochondrial genome of Carpodacus pulcherrimus (Passeriformes: Fringillidae).

The Himalayan Beautiful Rosefinch Carpodacus pulcherrimus, belongs to the family Fringillidae, distributed in central Himalayas from India (Himachal Pradesh) to southwest China and Bhutan. The conservation status of this species is least concern (LC) in IUCN. In this study, the complete mitogenome of C. pulcherrimus was determined. The mitogenome is a circular molecule of 16,797 bp in length, containing 13 protein-coding genes, 2 ribosome RNA genes, 22 transfer RNA genes, and 1 non-coding region. We reconstructed a phylogenetic tree based on Bayesian inference for other 14 Fringillidae species. The new mitogenome data would provide useful information for application in conservation genetics and further clarify phylogenetic evolution of this species.

The complete mitochondrial genome of Phoenicurus frontalis (Passeriformes: Muscicapidae).

The Blue-fronted Redstart Phoenicurus frontalis (Muscicapidae) belongs to the family Muscicapidae, distributed in central China, Qinghai-Tibet plateau and the Himalayas. The conservation status of this species is Least Concern (LC) in IUCN. In this study, the complete mitogenome of P. frontalis was determined. The mitogenome is a circular molecule of 16,776 bp in length, containing 13 protein-coding genes, 2 ribosome RNA genes, 22 transfer RNA genes, and 1 non-coding region. We reconstructed a phylogenetic tree based on Bayesian inference for 15 Passeriformes species. The new mitogenome data would provide useful information for application in conservation genetics and further clarify the phylogenetic evolution of this species.

Effects of aging on gene expression in blood of captive Tibetan macaques ( Macaca thibetana) and comparisons with expression in humans.

Changes in gene expression occur as animals, including primates, age. Macaques have long been used as a model species for primate evolution and biomedical studies. Here, to study gene expression in Tibetan macaques (Macaca thibetana, TMs) and its differences to humans, we applied RNA-Seq to obtain the blood transcriptomes of 24 TMs. In total, 2 523 age-associated differentially expressed genes (DEGs) were identified. Several pathways and processes that regulate aging, including the FoxO signaling pathway, autophagy, and platelet activation, were significantly enriched in the up-regulated DEGs. Two significantly age-related modules were identified by weighted gene co-expression network analysis (WGCNA). The TMs and humans shared 279 common DEGs, including 111 up-regulated and 141 down-regulated genes with advancing age in the same expression direction. However, 27 age-related DEGs presented the opposite expression direction in TMs as that in humans. For example, INPPL1, with inhibitory effects on the B cell receptor signaling pathway, was up-regulated in humans but down-regulated in TMs. In general, our study suggests that aging is a critical factor affecting gene expression in the captive TM population. The similarities and differences in gene expression patterns between TMs and humans could provide new insights into primate evolution and benefit TM model development.

Blood transcriptome analysis reveals gene expression features of breast-feeding rhesus macaque ( Macaca mulatta) infants.

During the breast-feeding period, infants undergo remarkable changes, including rapid physiological and developmental growth. However, little is known about gene expression features and sex-specific gene expression in breast-feeding infants. In this study, we sequenced 32 blood transcriptomes from 16 breast-feeding rhesus macaque ( Macaca mulatta) infants and their lactating mothers. We identified 218 differentially expressed genes (DEGs) between infants and mothers, including 91 up-regulated and 127 down-regulated DEGs in the infant group. Functional enrichment analysis of the up-regulated DEGs and unique hub genes in infants showed primary enrichment in immunity, growth, and development. Protein-protein interaction analysis also revealed that genes at key positions in infants were mainly related to development and immunity. However, we only detected 23 DEGs between female and male infants, including three DEGs located on chromosome X and 14 DEGs located on chromosome Y. Of these DEGs, TMF1 regulated nuclear protein 1 ( Trnp1), which was highly expressed in female infants, is crucial for controlling the tangential and radial expansion of the cerebral cortex in mammals. Thus, our study provides novel insight into the gene expression features of breast-feeding infants in non-human primates (NHPs) and reveals sex-specific gene expression between these infants.

Distribution patterns of microsatellites and development of its marker in different genomic regions of forest musk deer genome based on high throughput sequencing.

Forest musk deer (Moschus berezovskii, FMD) is an endangered artiodactyl species, male FMD produce musk. We have sequenced the whole genome of FMD, completed the genomic assembly and annotation, and performed bioinformatic analyses. Our results showed that microsatellites (SSRs) displayed nonrandomly distribution in genomic regions, and SSR abundances were much higher in the intronic and intergenic regions compared to other genomic regions. Tri- and hexanucleotide perfect (P) SSRs predominated in coding regions (CDSs), whereas, tetra- and pentanucleotide P-SSRs were less abundant. Trifold P-SSRs had more GC-contents in the 5'-untranslated regions (5'UTRs) and CDSs than other genomic regions, whereas mononucleotide P-SSRs had the least GC-contents. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs had different distributions in different genomic regions. The RCN of trinucleotide P-SSRs had increased significantly in the CDSs compared to the transposable elements (TEs), intronic and intergenic regions. The analysis of coefficient of variability (CV) of P-SSRs showed that the RCN of mononucleotide P-SSRs had relative higher variation in different genomic regions, followed by the CV pattern of RCN: dinucleotide P-SSRs > trinucleotide P-SSRs > tetranucleotide P-SSRs > pentanucleotide P-SSRs > hexanucleotide P-SSRs. The CV variations of RCN of the same mono- to hexanucleotide P-SSRs were relative higher in the intron and intergenic regions, followed by that in the TEs, and the relative lower was in the 5'UTR, CDSs and 3'UTRs. 58 novel polymorphic SSR loci were detected based on genotyping DNA from 36 captive FMD and 22 SSR markers finally showed polymorphism, stability, and repetition.

Identification and characterization of short tandem repeats in the Tibetan macaque genome based on resequencing data.

The Tibetan macaque, which is endemic to China, is currently listed as a Near Endangered primate species by the International Union for Conservation of Nature (IUCN). Short tandem repeats (STRs) refer to repetitive elements of genome sequence that range in length from 1-6 bp. They are found in many organisms and are widely applied in population genetic studies. To clarify the distribution characteristics of genome-wide STRs and understand their variation among Tibetan macaques, we conducted a genome-wide survey of STRs with next-generation sequencing of five macaque samples. A total of 1 077 790 perfect STRs were mined from our assembly, with an N50 of 4 966 bp. Mono-nucleotide repeats were the most abundant, followed by tetra- and di-nucleotide repeats. Analysis of GC content and repeats showed consistent results with other macaques. Furthermore, using STR analysis software (lobSTR), we found that the proportion of base pair deletions in the STRs was greater than that of insertions in the five Tibetan macaque individuals (P<0.05, t-test). We also found a greater number of homozygous STRs than heterozygous STRs (P<0.05, t-test), with the Emei and Jianyang Tibetan macaques showing more heterozygous loci than Huangshan Tibetan macaques. The proportion of insertions and mean variation of alleles in the Emei and Jianyang individuals were slightly higher than those in the Huangshan individuals, thus revealing differences in STR allele size between the two populations. The polymorphic STR loci identified based on the reference genome showed good amplification efficiency and could be used to study population genetics in Tibetan macaques. The neighbor-joining tree classified the five macaques into two different branches according to their geographical origin, indicating high genetic differentiation between the Huangshan and Sichuan populations. We elucidated the distribution characteristics of STRs in the Tibetan macaque genome and provided an effective method for screening polymorphic STRs. Our results also lay a foundation for future genetic variation studies of macaques.

Distribution patterns and variation analysis of simple sequence repeats in different genomic regions of bovid genomes.

As the first examination of distribution, guanine-cytosine (GC) pattern, and variation analysis of microsatellites (SSRs) in different genomic regions of six bovid species, SSRs displayed nonrandomly distribution in different regions. SSR abundances are much higher in the introns, transposable elements (TEs), and intergenic regions compared to the 3'-untranslated regions (3'UTRs), 5'UTRs and coding regions. Trinucleotide perfect SSRs (P-SSRs) were the most frequent in the coding regions, whereas, mononucleotide P-SSRs were the most in the introns, 3'UTRs, TEs, and intergenic regions. Trifold P-SSRs had more GC-contents in the 5'UTRs and coding regions than that in the introns, 3'UTRs, TEs, and intergenic regions, whereas mononucleotide P-SSRs had the least GC-contents in all genomic regions. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs showed significantly different distributions in different regions (P < 0.01). Except for the coding regions, mononucleotide P-SSRs had the most RCNs, followed by the pattern: di- > tri- > tetra- > penta- > hexanucleotide P-SSRs in the same regions. The analysis of coefficient of variability (CV) of SSRs showed that the CV variations of RCN of the same mono- to hexanucleotide SSRs were relative higher in the intronic and intergenic regions, followed by the CV variation of RCN in the TEs, and the relative lower was in the 5'UTRs, 3'UTRs, and coding regions. Wide SSR analysis of different genomic regions has helped to reveal biological significances of their distributions.

Reassessment of the taxonomic status of Craseomys and three controversial species of Myodes and Alticola (Rodentia: Arvicolinae).

The genera Myodes (red-backed voles) and Alticola (mountain voles) appear to be sister taxa based on morphological similarities, but molecular analyses fail to resolve them as monophyletic genera owing to the uncertain taxonomic status of Craseomys and Phaulomys. As a result of incomplete sampling of related specimens, ongoing controversies on the taxonomic positions of several generic and specific taxa necessitate further clarifications. Herein, we combined molecular, morphometric, and geometric morphometric approaches to analyze 217 specimens of 10 taxa of Myodes and Alticola systematically. We sequenced three genes (Cytb, COI, GHR) de novo from specimens with fresh tissues, and published sequences for M. shanseius and A. stoliczkanus for the first time. Based on this new molecular dataset, we produced phylogenetic trees using Bayesian inference, maximum likelihood, and maximum parsimony approaches. Our molecular and morphological analyses both identified three primary clades within Myodes and Alticola. The Craseomys-Phaulomys clade consistently separated from Myodes sensu stricto (s. str.) and Alticola s. str.-Platycranius. Our results support the resurrection of the genus Craseomys and the treatment of Phaulomys as its junior synonym. As Craseomys shanseius clustered with C. rufocanus in three gene phylogenies and this assessment was congruent with morphological results, we assigned C. shanseius to a subspecies of C. rufocanus. Specimens from one sampling site in Pulan County of Tibet possess M3 patterns typical of A. stoliczkanus and A. stracheyi, despite clustering together in matrilineal genealogy. Thus, we tentatively assigned A. stracheyi as a junior synonym of A. stoliczkanus. Our analyses confirmed the validity of A. semicanus and unambiguously distinguished it from A. argentatus by the ratio of tail length to head-body length, color of tail and feet, M3 pattern, and distribution.

Comparison of microsatellite distribution in genomes of Centruroides exilicauda and Mesobuthus martensii.

In this study, we characterized the distribution of microsatellites in the genomes and genes of Centruroides exilicauda and Mesobuthus martensii, carried out Gene Ontology (GO) analysis and GO enrichment analysis of coding sequences (CDSs) with microsatellite (SSR). In addition, over-represented GO functions related to environmental interactions, development process and methylation were identified to develop functional markers and facilitate further analysis of microsatellite function in the genes of scorpions. Location analysis indicated that microsatellites were predominantly concentrated at both ends of genes. Most genes containing microsatellite had the SSR present at only one locus, from which we infer that the number of SSRs per gene is limited even though intragenic tandem repeats can generate functional variability. Lastly, we identified 75 SSRs in 64 genes of 54 expanded gene families and 1 SSR in the toxin gene of Mesobuthus martensii, allowing future studies on the effect of microsatellites on gene function.

Distinct patterns of simple sequence repeats and GC distribution in intragenic and intergenic regions of primate genomes.

As the first systematic examination of simple sequence repeats (SSRs) and guanine-cytosine (GC) distribution in intragenic and intergenic regions of ten primates, our study showed that SSRs and GC displayed nonrandom distribution for both intragenic and intergenic regions, suggesting that they have potential roles in transcriptional or translational regulation. Our results suggest that the majority of SSRs are distributed in non-coding regions, such as the introns, TEs, and intergenic regions. In these primates, trinucleotide perfect (P) SSRs were the most abundant repeats type in the 5'UTRs and CDSs, whereas, mononucleotide P-SSRs were the most in the intron, 3'UTRs, TEs, and intergenic regions. The GC-contents varied greatly among different intragenic and intergenic regions: 5'UTRs > CDSs > 3'UTRs > TEs > introns > intergenic regions, and high GC-content was frequently distributed in exon-rich regions. Our results also showed that in the same intragenic and intergenic regions, the distribution of GC-contents were great similarity in the different primates. Tri- and hexanucleotide P-SSRs had the most GC-contents in the 5'UTRs and CDSs, whereas mononucleotide P-SSRs had the least GC-contents in the six genomic regions of these primates. The most frequent motifs for different length varied obviously with the different genomic regions.

[Enrichment of giant panda microsatellite markers using dynal magnet beads].

The 400 -600 bp DNA fractions of giant panda containing STR sequences were captured by hybridization with the oligonucleotide probes attached to streptavadin coated magnetic beads (Dynal). The enriched DNA were ligated into pGEM-T and then transformed into E. coil JM109 competent cells. In total 260 positive clones were identified from 2 880 transformants in the libraries which were screened by gamma-32 P radiolabelled probes. Finally, we got 54 sequences and successfully designed 37 pairs of STR primers for giant panda. The results showed that this method is very efficient to isolate microsatellite markers.

[Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.