Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds.

Copy number polymorphisms (CNPs) of antimicrobial peptides (AMPs) in livestock can influence the innate immune response of individuals. We conducted a high-resolution analysis of the genomic variations of porcine cathelicidin PR39 using cloned PR39 amplicons corresponding to the 5' untranslated region (UTR) to 3' UTR from four individuals of three different pig breeds. We identified 15 different sequences corresponding to 9 different coding domain sequences (CDSs), encoding 7 different protein sequences consisting of 3 functional and 4 non-functional forms. Subsequently, we developed a PR39 CNP typing method using real-time polymerase chain reaction (PCR) and analyzed the PR39 copy numbers from 44 pigs of six breeds. Significant variations in PR39 copies ranging from 2 to 10 copies, with a mean copy number of 5, were observed among all commercial breeds, except the wild boar. Among the different breeds, the PR39 copy number was highest (10) in Korean native pigs. Gene expression analysis showed that PR39 expression was correlated with the copy number. Moreover, the comparative analysis of the cathelicidin cluster-containing region among eight mammalian species showed the complete evolutionary conservation of the region, except for differences in the degree of cathelicidin expansion in each species. Therefore, characterization of CNPs in AMP genes could aid in improving the genetic potential of innate immune responses in livestock animals.

Protection of intestinal immune barrier against ischemia/reperfusion injury in a swine model using anisodamine hydrobromide

Abstract Intestinal ischemia/reperfusion (I/R) causes barrier impairment and bacterial influx. This study explored the protective effects of anisodamine hydrobromide (AH) on intestinal I/R injury caused by cardiopulmonary resuscitation (CPR) after cardiac arrest (CA). After successful CPR, minipigs were randomly divided into two groups (n = 8): saline and AH (4 mg/kg), and then treated with saline or AH via central venous injection, respectively. The same procedures without ventricular fibrillation initiation were conducted in the Sham group (n = 8). Levels of interferon gamma (IFN-γ) and interleukin 4 (IL-4) were measured at different time points (0, 0.5, 1, 2, 4, and 6 h) in serum and 6 h in gut associated lymphoid tissues (GALTs) after the return of spontaneous circulation (ROSC) to evaluate changes in the proportion of T-helper type 1 (Th1) and T-helper type 2 (Th2). Moreover, the positive culture rates of GALTs were examined to evaluate bacterial translocation. AH treatment markedly alleviated aberrant arterial blood gas and hemodynamics as well as intestinal macroscopic and morphological changes after CPR. Moreover, AH treatment significantly increased IFN-γ and decreased IL-4 in both serum and GALTs. Furthermore, AH treatment dramatically decreased positive bacterial growth in GALTs. AH treatment mitigated immunosuppression caused by intestinal I/R and protected the intestinal immune barrier against bacterial translocation, thereby reducing the risk of secondary intestinal infection

Detecting the selection signatures in Chinese Duroc,Landrace, Yorkshire, Liangshan, and Qingyu pigs.

Here we used two kinds of chips data from 5 pig breeds, Chinese Duroc (DD), Landrace (LL), Yorkshire (YY), Liangshan (LS), and Qingyu pigs (QY) in China to identify genes which show evidence of selection during domestication. Four breed pairs, LS-YY, QY-YY, DD-YY, and LL-YY pair, were performed to detect selection signatures using the Fst method. Then we identified a list of genes that played key roles in domestication and artificial selection. For example, the PTPRM gene was shared in LS-YY, QY-YY, and DD-YY pairs and it regulates a variety of cellular processes including cell growth, differentiation as signaling molecules. The HACD3 gene was shared in QY-YY and DD-YY pairs, and the HACD3 protein is involved in the production of very long-chain fatty acids of different chain lengths. Besides, the MYH11 gene that related to muscle contraction was found in LS-YY and LL-YY pair. These results suggested that genes related to immunity, disease resistance, and metabolism were subjected to strong selection pressure in Chinese domestic pigs in the progress of domestication and evolution; however, genes related to appearance, production performance, and reproduction were undergone strong artificial selection in commercial pig breeds.

Genetic architecture and major genes for backfat thickness in pig lines of diverse genetic backgrounds.

BACKGROUND: Backfat thickness is an important carcass composition trait for pork production and is commonly included in swine breeding programmes. In this paper, we report the results of a large genome-wide association study for backfat thickness using data from eight lines of diverse genetic backgrounds. METHODS: Data comprised 275,590 pigs from eight lines with diverse genetic backgrounds (breeds included Large White, Landrace, Pietrain, Hampshire, Duroc, and synthetic lines) genotyped and imputed for 71,324 single-nucleotide polymorphisms (SNPs). For each line, we estimated SNP associations using a univariate linear mixed model that accounted for genomic relationships. SNPs with significant associations were identified using a threshold of p < 10-6 and used to define genomic regions of interest. The proportion of genetic variance explained by a genomic region was estimated using a ridge regression model. RESULTS: We found significant associations with backfat thickness for 264 SNPs across 27 genomic regions. Six genomic regions were detected in three or more lines. The average estimate of the SNP-based heritability was 0.48, with estimates by line ranging from 0.30 to 0.58. The genomic regions jointly explained from 3.2 to 19.5% of the additive genetic variance of backfat thickness within a line. Individual genomic regions explained up to 8.0% of the additive genetic variance of backfat thickness within a line. Some of these 27 genomic regions also explained up to 1.6% of the additive genetic variance in lines for which the genomic region was not statistically significant. We identified 64 candidate genes with annotated functions that can be related to fat metabolism, including well-studied genes such as MC4R, IGF2, and LEPR, and more novel candidate genes such as DHCR7, FGF23, MEDAG, DGKI, and PTN. CONCLUSIONS: Our results confirm the polygenic architecture of backfat thickness and the role of genes involved in energy homeostasis, adipogenesis, fatty acid metabolism, and insulin signalling pathways for fat deposition in pigs. The results also suggest that several less well-understood metabolic pathways contribute to backfat development, such as those of phosphate, calcium, and vitamin D homeostasis.

Chemical Characteristics of Croatian Traditional Istarski prsut (PDO) Produced from Two Different Pig Genotypes.

Chemical characteristics of raw and processed Istarski prsut (PDO) produced from two different pig genotypes were studied with special emphasis on amino and fatty acid composition and factors of lipid stability. Raw hams of Large White (LW)xLandrace (L), and (LWxL)xDuroc (D) pig genotypes were used in the study (20 hams of each genotype). All left raw hams from each carcass were processed in accordance with the PDO specification of Istarski prsut, and other half (the right ones) of LWxL)xD genotype were used for analyses of raw hams (fresh muscles). Istarski prsut was evaluated on the basis of the chemical parameters of the raw and matured lean ham. The process of dry curing significantly influenced the chemical properties of Istarski prsut. Despite the higher content of intramuscular fat and polyunsaturated fatty acids, the fat of (LWxL)xD ham was much more resistant to hydrolysis and oxidation, suggesting that fatty acid profile and other factors, also play a significant role. Significant differences between pig genotypes in the amino acid and fatty acid profiles were found. The analyzed Istarski prsut may be distinguished by prints of multivariate chemometric statistical analysis, based on their amino acid and fatty acid compositions.

Microsatellite DNA Analysis for Diversity Study, Individual Identification and Parentage Control in Pig Breeds in Poland.

Swine DNA profiling is of high importance for animal identification and parentage verification. The aim of this study was to test a set of 14 microsatellite (STR) markers recommended by ISAG for parentage verification in Polish Landrace (PL, n = 900), Polish Large White (PLW, n = 482), Pulawska (PUL, n = 127), and Duroc pigs (DU n = 108). The studied breeds showed a medium level of genetic differentiation. The average value of heterozygosity and degree of polymorphism (PIC) were above 0.5 for the studied breeds, except for the DU breed (PIC = 0.477). The population inbreeding coefficient indicates an absence of inbreeding in the studied breeds (an average value of FIS = 0.007). The cumulative power of discrimination for all breeds reached high values close to 1.0, while the probability of identity (PID) was low, with PID values ranging between 10-9 (for DU) and 10-12 (for PLW). The cumulative exclusion probability for PE1 and PE2 showed that the parentage can be confirmed with a probability of from 92.75% to 99.01% and from 99.49% to 99.97%, respectively.

Pygmy hogs.

Manon de Visser and colleagues introduce the rarest and smallest wild pig species, the pygmy hog (Porcula salvania).

Screening out important substances for distinguishing Chinese indigenous pork and hybrid pork and identifying different pork muscles by analyzing the fatty acid and nucleotide contents.

Fatty acids and nucleotides are major contributors of pork flavor, which are key quality traits influencing consumers' preference. In this study, we investigated the contents of nucleotides (inosinic acid, inosine, and hypoxanthine) and 26 fatty acids by high-performance liquid chromatography and gas chromatography in longissimus dorsi and semimembranous muscle of four breeds (Dongbei Min pig, Beijing Black pig, Wuzhishan pig, and DLY pigs). Combining with multivariate statistics, we found significant differences in the nucleotides and fatty acids compositions among the breeds and muscles. Furthermore, the content of total unsaturated fatty acids and DHA were higher in native pigs than crossbred pigs. This study provides a theoretical basis for understanding the composition of flavor precursor substances, as well as for the identification of different muscles.

A gene co-association network regulating gut microbial communities in a Duroc pig population.

BACKGROUND: Analyses of gut microbiome composition in livestock species have shown its potential to contribute to the regulation of complex phenotypes. However, little is known about the host genetic control over the gut microbial communities. In pigs, previous studies are based on classical "single-gene-single-trait" approaches and have evaluated the role of host genome controlling gut prokaryote and eukaryote communities separately. RESULTS: In order to determine the ability of the host genome to control the diversity and composition of microbial communities in healthy pigs, we undertook genome-wide association studies (GWAS) for 39 microbial phenotypes that included 2 diversity indexes, and the relative abundance of 31 bacterial and six commensal protist genera in 390 pigs genotyped for 70 K SNPs. The GWAS results were processed through a 3-step analytical pipeline comprised of (1) association weight matrix; (2) regulatory impact factor; and (3) partial correlation and information theory. The inferred gene regulatory network comprised 3561 genes (within a 5 kb distance from a relevant SNP-P < 0.05) and 738,913 connections (SNP-to-SNP co-associations). Our findings highlight the complexity and polygenic nature of the pig gut microbial ecosystem. Prominent within the network were 5 regulators, PRDM15, STAT1, ssc-mir-371, SOX9 and RUNX2 which gathered 942, 607, 588, 284 and 273 connections, respectively. PRDM15 modulates the transcription of upstream regulators of WNT and MAPK-ERK signaling to safeguard naive pluripotency and regulates the production of Th1- and Th2-type immune response. The signal transducer STAT1 has long been associated with immune processes and was recently identified as a potential regulator of vaccine response to porcine reproductive and respiratory syndrome. The list of regulators was enriched for immune-related pathways, and the list of predicted targets includes candidate genes previously reported as associated with microbiota profile in pigs, mice and human, such as SLIT3, SLC39A8, NOS1, IL1R2, DAB1, TOX3, SPP1, THSD7B, ELF2, PIANP, A2ML1, and IFNAR1. Moreover, we show the existence of host-genetic variants jointly associated with the relative abundance of butyrate producer bacteria and host performance. CONCLUSIONS: Taken together, our results identified regulators, candidate genes, and mechanisms linked with microbiome modulation by the host. They further highlight the value of the proposed analytical pipeline to exploit pleiotropy and the crosstalk between bacteria and protists as significant contributors to host-microbiome interactions and identify genetic markers and candidate genes that can be incorporated in breeding program to improve host-performance and microbial traits. Video Abstract.

A 14 bp indel polymorphism in the promoter region is associated with different responses to porcine circovirus type 2 infection by regulating MRC1 transcription.

Mannose receptor, C type 1 (MRC1) is a key factor in regulating the body's immune response to resist pathogen invasions. In this study, mRNA expressions of MRC1 gene in nine porcine organs/tissues were compared between Laiwu (LW) and Yorkshire × Landrace crossbred (YL) pigs prior to and post PCV2 infection. We found that, for pigs uninfected with PCV2, MRC1 mRNA expressions in the lung, spleen, large intestine, small intestine and mesenteric lymph node tissues of LW were significantly higher than those of YL pigs (P < 0.05). After PCV2 infection, MRC1 mRNA levels in the liver, kidney and mesenteric lymph node were significantly increased in LW pigs (P < 0.05); while, significantly decreased in the heart and lung tissues of YL pigs (P < 0.05). The transcriptional activity of porcine MRC1 promoter was further analyzed to investigate the molecular mechanism underlying these expressional differences in response to PCV2 infection. Luciferase assay indicated that a 14 bp indel polymorphism "GTTTTTTTTTTTTT" at the site -864 of MRC1 promoter contributed to the transcriptional activity. The frequency of 14 bp insertion in LW and Dapulian pigs, generally resistant to PCV2 infection, was higher than that in Duroc, Landrace and Yorkshire pigs, which were sensitive to PCV2 infection. The promoter with 14 bp insertion displayed higher MRC1 transcription level both prior to and post PCV2 infection compared with that carrying no insertion in PK15 cells (P < 0.01). The results suggest that this 14 bp indel polymorphism is associated with different responses to PCV2 infection by regulating MRC1 transcription.

Maternal Transmission Ratio Distortion in Two Iberian Pig Varieties.

Transmission ratio distortion (TRD) is defined as the allele transmission deviation from the heterozygous parent to the offspring from the expected Mendelian genotypic frequencies. Although TRD can be a confounding factor in genetic mapping studies, this phenomenon remains mostly unknown in pigs, particularly in traditional breeds (i.e., the Iberian pig). We aimed to describe the maternal TRD prevalence and its genomic distribution in two Iberian varieties. Genotypes from a total of 247 families (dam and offspring) of Entrepelado (n = 129) and Retinto (n = 118) Iberian varieties were analyzed. The offspring were sired by both ungenotyped purebred Retinto and Entrepelado Iberian boars, regardless of the dam variety used. After quality control, 16,246 single-nucleotide polymorphisms (SNPs) in the Entrepelado variety and 9744 SNPs in the Retinto variety were analyzed. Maternal TRD was evaluated by a likelihood ratio test under SNP-by-SNP, adapting a previous model solved by Bayesian inference. Results provided 68 maternal TRD loci (TRDLs) in the Entrepelado variety and 24 in the Retinto variety (q < 0.05), with mostly negative TRD values, increasing the transmission of the minor allele. In addition, both varieties shared ten common TRDLs. No strong evidence of biological effects was found in genes with TRDLs. However, some biological processes could be affected by TRDLs, such as embryogenesis at different levels and lipid metabolism. These findings could provide useful insight into the genetic mechanisms to improve the swine industry, particularly in traditional breeds.

Gut microbiome composition differences among breeds impact feed efficiency in swine.

BACKGROUND: Feed efficiency is a crucial parameter in swine production, given both its economic and environmental impact. The gut microbiota plays an essential role in nutrient digestibility and is, therefore, likely to affect feed efficiency. This study aimed to characterize feed efficiency, fatness traits, and gut microbiome composition in three major breeds of domesticated swine and investigate a possible link between feed efficiency and gut microbiota composition. RESULTS: Average daily feed intake (ADFI), average daily gain (ADG), feed conversion ratio (FCR), residual feed intake (RFI), backfat, loin depth, and intramuscular fat of 615 pigs belonging to the Duroc (DR), Landrace (LR), and Large White (LW) breeds were measured. Gut microbiota composition was characterized by 16S rRNA gene sequencing. Orthogonal contrasts between paternal line (DR) and maternal lines (LR+LW) and between the two maternal lines (LR versus LW) were performed. Average daily feed intake and ADG were statistically different with DR having lower ADFI and ADG compared to LR and LW. Landrace and LW had a similar ADG and RFI, with higher ADFI and FCR for LW. Alpha diversity was higher in the fecal microbial communities of LR pigs than in those of DR and LW pigs for all time points considered. Duroc communities had significantly higher proportional representation of the Catenibacterium and Clostridium genera compared to LR and LW, while LR pigs had significantly higher proportions of Bacteroides than LW for all time points considered. Amplicon sequence variants from multiple genera (including Anaerovibrio, Bacteroides, Blautia, Clostridium, Dorea, Eubacterium, Faecalibacterium, Lactobacillus, Oscillibacter, and Ruminococcus) were found to be significantly associated with feed efficiency, regardless of the time point considered. CONCLUSIONS: In this study, we characterized differences in the composition of the fecal microbiota of three commercially relevant breeds of swine, both over time and between breeds. Correlations between different microbiome compositions and feed efficiency were established. This suggests that the microbial community may contribute to shaping host productive parameters. Moreover, our study provides important insights into how the intestinal microbial community might influence host energy harvesting capacity. A deeper understanding of this process may allow us to modulate the gut microbiome in order to raise more efficient animals. Video Abstract.

Identification of a novel premature stop codon and other recurrent variations in the porcine gelsolin gene.

Gelsolin is an actin-binding protein that plays a significant role in sustaining cell motility and cell metabolism. Investigations of the mutations present in the key regions of gelsolin provide extensive information to further understand the mechanism by which gelsolin causes variation in the phenotype [e.g., residual feed intake (RFI) or feed efficiency ]of pigs. However, there have been no investigations of the variation in functional binding regions or research on Chinese native pigs. In this study, three key regions of gelsolin were investigated in 144 pigs from six breeds using a sequencing method. The results revealed 16 nucleotide substitutions, eight of which (c.42-13G/T, c.59 T/C, c.86C/T, c.87G/T, c.104C/T, c.144 T/C, c.206G/C, and c.237 + 21A/G) were novel and identified in intron 1, exon 2, and intron 2. Two variants (c.87G/T and c.144 T/C) resulted in a premature stop codon (p.Gly16Uga(Stop)) and an amino acid change (p.Tyr35His), respectively. In region 1, c.144 T/C was the most common (at a total frequency of 46.5%), followed by c.42-13G/T (at a total frequency of 41.7%). In region 2, two variants (c.350A/G and c.374A/G) were most common (both at a total frequency of 36.1%). There were significant differences (P < 0.05) in variant frequencies between Chinese indigenous pigs and overseas pigs. Our findings revealed one novel premature stop codon and eight novel variations in re-sequencing regions, which suggest that these variations of gelsolin may influence its mRNA expression and consequently affect production traits in swine.

Discovery of significant porcine SNPs for swine breed identification by a hybrid of information gain, genetic algorithm, and frequency feature selection technique.

BACKGROUND: The number of porcine Single Nucleotide Polymorphisms (SNPs) used in genetic association studies is very large, suitable for statistical testing. However, in breed classification problem, one needs to have a much smaller porcine-classifying SNPs (PCSNPs) set that could accurately classify pigs into different breeds. This study attempted to find such PCSNPs by using several combinations of feature selection and classification methods. We experimented with different combinations of feature selection methods including information gain, conventional as well as modified genetic algorithms, and our developed frequency feature selection method in combination with a common classification method, Support Vector Machine, to evaluate the method's performance. Experiments were conducted on a comprehensive data set containing SNPs from native pigs from America, Europe, Africa, and Asia including Chinese breeds, Vietnamese breeds, and hybrid breeds from Thailand. RESULTS: The best combination of feature selection methods-information gain, modified genetic algorithm, and frequency feature selection hybrid-was able to reduce the number of possible PCSNPs to only 1.62% (164 PCSNPs) of the total number of SNPs (10,210 SNPs) while maintaining a high classification accuracy (95.12%). Moreover, the near-identical performance of this PCSNPs set to those of bigger data sets as well as even the entire data set. Moreover, most PCSNPs were well-matched to a set of 94 genes in the PANTHER pathway, conforming to a suggestion by the Porcine Genomic Sequencing Initiative. CONCLUSIONS: The best hybrid method truly provided a sufficiently small number of porcine SNPs that accurately classified swine breeds.

Comparative Transcriptomic Analysis of Subcutaneous Adipose Tissue from Local Pig Breeds.

When compared to modern lean-type breeds, Portuguese local Alentejano (AL) and Bísaro (BI) pig breeds present a high potential for subcutaneous and intramuscular fat (IMF) deposition which contributes for better meat quality. The aim of this work was to explore the genome function to better understand the underlying physiological mechanisms associated with body fat accretion. Dorsal subcutaneous fat samples were collected at slaughter from adult animals (n = 4 for each breed) with ~150 kg body weight. Total RNA was obtained and sequenced for transcriptome analysis using DESeq2. A total of 458 differentially expressed (DE) genes (q-value < 0.05) were identified, with 263 overexpressed in AL and 195 in BI. Key genes involved in de novo fatty acid biosynthesis, elongation and desaturation were upregulated in AL such as ACLY, FASN, ME1, ELOVL6 and SCD. A functional enrichment analysis of the DE genes was performed using Ingenuity Pathway Analysis. Cholesterol synthesis is suggested to be higher in AL via SREBF2, SCAP and PPARG, while lipolytic activity may be more active in BI through GH and AMPK signalling. Increased signalling of CD40 together with the predicted activation of INSIG1 and INSIG2 in BI suggests that this breed is more sensitive to insulin whereas the AL is less sensitive like the Iberian breed.

Function of CCL5 in maternal-fetal interface of pig during early pregnancy.

Chemokines refer to chemoattractant cytokines, which have crucial functions in inflammation and immune responses in multiple cellular processes. In the present study, we described the potential role of porcine CCL5 in embryo implantation and fetal-maternal environment during early pregnancy. We first carried out phylogenetic analysis of porcine CCL5, and analyzed the cell specific localization of CCL5 and its receptor CCR3 in a kinetic approach within porcine estrous cycles and early gestation stage. In addition, CCL5 stimulated porcine uterine luminal epithelial (pLE) and porcine trophectoderm (pTr) cell proliferations, and cell cycle progressions via AKT and MAPK intracellular signaling tractions. Furthermore, CCL5 attenuated tunicamycin-induced endoplasmic reticulum (ER) stress signaling, and lipopolysaccharides-triggered inflammatory responses in pLE and pTr cells. Taken together, our study showed that CCL5 is involved in the placental development or promotes the placental development.

Suidae from Kanapoi.

The whole collection of Suidae from Kanapoi is revised in the context of the systematics and evolution of Nyanzachoerus in the Pliocene of Eastern Africa. It contains only two species, Nyanzachoerus kanamensis and Notochoerus jaegeri. The size and morphology of their premolars overlap, but not those of their m3s. No transitional form between them is known in Kenya, but some populations from Uganda and Ethiopia display intermediate characters, suggesting that No. jaegeri could be descended from a kanamensis-like ancestor. However, the cranial remains of No. jaegeri from Kanapoi are insufficient to formally establish the affinities of the species. On the basis of the dentition, Notochoerus euilus could be descended from No. jaegeri. The noticeable absence of Kolpochoerus at Kanapoi (and in the whole Turkana Basin at that time) remains unexplained. The presence of a species with affinity to Nyanzachoerus tulotos at Ekora raises the possibility that uppermost Miocene sediments occur there.

First report of Sus strozzii (Suidae, Mammalia) from the Early Pleistocene of Hungary (Dunaalmás) and species distinction based on deciduous teeth.

Herein we describe a fragment of a mandible with a deciduous fourth premolar (dp4) from the Early Pleistocene locality of Dunaalmás, representing the first confirmed report of Sus strozzii from Hungary. The comparison of dp4 measurements supports a statistically significant distinction between S. strozzii and Sus scrofa. The two species overlap in time during the late Early Pleistocene of Europe (Epivillafranchian), but suid remains of this time-span are seldom classified at a species level. The correct taxonomic identification of the Epivillafranchian suids, which are often associated with evidences of hominin presence, is of great palaeoenvironmental value because S. scrofa and S. strozzii possess different ecological requirements.

Use of a non-destructive sampling method for characterization of Iberian cured ham breed and feeding regime using GC-IMS.

Iberian cured ham from purebred pigs fattened on grazing acorns is a highly appreciated product. There are several analytical methods to avoid its labelling fraud; however, these require opening the ham. The aim of this work is testing a non-destructive sampling method which does not spoil the physical integrity of the ham. It consists of a puncture with a needle imitating a traditional olfactory system. After removing the needle impregnated with fat, its volatiles are analyzed with a Gas Chromatography (GC) coupled to Ion Mobility Spectrometry (IMS). The potential of this methodology was studied analyzing 156 Iberian hams from pigs under two different feeding regimes (acorns versus feed) and from different breed (Iberian versus Duroc crossed). Intensity of GC-IMS plot features was extracted and chemometric differentiation models were obtained; one for feeding regime and another for breed, providing validated classification rates of 91.7% and 100%, respectively. In addition, 29 features used for construction of both models were tentatively identified using chemical standards. The suitability of the method for quality control analysis was characterized by means of a precision study. As a conclusion, GC-IMS becomes a useful tool to guarantee dry-cured Iberian ham authenticity and detect labelling fraud.