Efficiently Summarizing Relationships in Large Samples: A General Duality Between Statistics of Genealogies and Genomes

Efficiently Summarizing Relationships in Large Samples: A General Duality Between Statistics of Genealogies and Genomes

Is the mitochondrial DNA (mtDNA) copy quantity of cumulus granulosa cells (CGCs) associated to the maturation of oocyte cytoplasm?Compared with the mtDNA copy quantity of CGCs from germinal vesicles (GV), CGCs from Metaphase I (MI) oocytes seem to have a decrease mtDNA copy quantity.The development and growth of CGCs and oocyte are synchronised.

The interplay between CGCs and the oocyte supplies the suitable stability of vitality, which is important for mammalian oocyte growth. Moreover, in the oocyte-cumulus advanced (OCC), mature oocytes with larger mtDNA copy numbers are likely to have corresponding CGCs with larger mtDNA copy numbers.

This is a potential examine of 302 OCCs obtained from 70 girls present process in vitro fertilisation with intracytoplasmic sperm injection (ICSI) on the Reproductive and Genetic Hospital of CITIC-Xiangya, between 24 February 2018 and 21 December 2019. The CGCs have been divided into three teams (GV, MI and MII phases) based mostly on the maturation standing of their corresponding oocyte.

The pattern sizes (n = 302) of CGCs in the three phases have been 63 (CGCGV), 70 (CGCMI) and 169 (CGCMII), respectively. Some of the samples (n = 257) was used to quantify the mtDNA copy quantity, whereas the remainder (n = 45) have been used to analyse the expression stage of mitochondrial genes. Furthermore, we retrieved 82 immature oocytes from among the many 257 OCCs used for mtDNA copy numbers, together with 36 GV oocytes and 46 MI oocytes, for evaluation of oocyte mtDNA.

We chosen genes with excessive consistency of real-time PCR outcomes to precisely measure the mtDNA copy quantity by testing the efficacy and the reproducibility in entire genome amplification (WGA) samples from a human embryonic stem cell line. The CGCs of every oocyte have been individually remoted.

The mtDNA copy quantity and gene expression of the CGCs have been assessed utilizing real-time PCR methods. Mitochondrial DNA copy quantity of the corresponding immature oocytes was additionally evaluated.MT-ND1, MT-CO1 and β-globin genes have been chosen for the evaluation of mtDNA content material, and mRNA expressions of MT-ND1, MT-CO1, PGC-1α and TFAM have been additionally measured. The genome of 257 CGCs and 82 immature oocytes have been amplified based on the a number of displacement amplification (MDA) protocol, and RNA was extracted from 45 CGCs.

Compared with CGCGV, CGCMI had a considerably decrease mtDNA copy quantity. In the MT-ND1 assay, the CGCGV: CGCMI was [270 ± 302]: [134 ± 201], P = 0.015. In the MT-CO1 assay, CGCGV: CGCMI was [205 ± 228]: [92 ± 112], P = 0.026. There was no statistical distinction in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [270 ± 302]: [175 ± 223], P = 0.074. In the MT-CO1 assay, CGCGV: CGCMII was [205 ± 228]: [119 ± 192], P = 0.077.

Efficiently Summarizing Relationships in Large Samples: A General Duality Between Statistics of Genealogies and Genomes
Efficiently Summarizing Relationships in Large Samples: A General Duality Between Statistics of Genealogies and Genomes

No statistical distinction of mtDNA copy quantity was noticed between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [134 ± 201]: [175 ± 223], P = 0.422. In the MT-CO1 assay, CGCMI: CGCMII was [92 ± 112]: [119 ± 192], P = 0.478. To confirm the reliability of the above outcomes, we additional analysed the mtDNA copy quantity of CGCs of 14 sufferers with GV, MI and MII oocytes, and the outcomes confirmed that the mtDNA copy quantity of CGCMI could also be decrease.

The mtDNA copy quantity of CGCGV and CGCMI was statistically totally different in the MT-ND1 assay the place CGCGV: CGCMI was [249 ± 173]: [118 ± 113], P = 0.016, however in the MT-CO1 assay, CGCGV: CGCMI was [208 ± 199]: [83 ± 98], P = 0.109. There was no important distinction in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [249 ± 173]: [185 ± 200], P = 0.096. In the MT-CO1 assay, CGCGV: CGCMII was [208 ± 199]: [114 ± 139], P = 0.096.

There was additionally no important distinction in mtDNA between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [118 ± 113]: [185 ± 200], P = 0.198. In the MT-CO1 assay, CGCMI: CGCMII was [83 ± 98]: [114 ± 139], P = 0.470. Moreover, there have been no statistical variations in the expression ranges of MT-ND1, MT-CO1, PGC-1α and TFAM between CGCGV, CGCMI and CGCMII (P > 0.05).N/A.Due to the moral points, the examine didn’t quantify the mtDNA content material of MII oocytes.

Thus, whether or not the change in mtDNA copy quantity in CGCs is said to the totally different developmental phases of oocytes has not been additional confirmed. Moreover, the pattern measurement was comparatively small.The mtDNA copy quantity of CGCs decreases from the GV part to the MI part and stays regular from the MI to MII stage.

At totally different phases of oocyte maturation, the mtDNA of CGCs might bear self-degradation and replication to satisfy the vitality necessities of the corresponding oocyte and the maturation of the oocyte cytoplasm.Funding was offered by the National Key R&D Program of China (Grant 2018YFC1003100, to L.H.), the science and know-how main challenge of the Ministry of Science and Technology of Hunan Province, China (grant 2017SK1030, to G.L.), the National Natural Science Foundation of China (grant 81873478, to L.H.), and Merck Serono China Research Fund for Fertility Experts (to L.H.). There isn’t any battle of curiosity.

Phyloanatomic characterization of the distinct T cell and monocyte contributions to the peripheral blood HIV inhabitants throughout the host

Human immunodeficiency virus (HIV) is a quickly evolving virus, permitting its genetic sequence to behave as a fingerprint for epidemiological processes amongst, in addition to inside, particular person contaminated hosts.

Though primarily infecting the CD4+ T-cell inhabitants, HIV will also be discovered in monocytes, an immune cell inhabitants that differs in a number of features from the canonical T-cell viral goal. Using single genome viral sequencing and statistical phylogenetic inference, we investigated the viral RNA range and relative contribution of every of these immune cell sorts to the viral inhabitants throughout the peripheral blood.

Results present proof of an elevated prevalence of circulating monocytes harboring virus in people with excessive viral load in the absence of suppressive antiretroviral remedy. Bayesian phyloanatomic evaluation of three of these people demonstrated a measurable position for these cells, however not the circulating T-cell inhabitants, as a supply of cell-free virus in the plasma, supporting the speculation that these cells can act as a further conduit of virus unfold.

A major genetic determinant of autoimmune diseases is associated with the presence of autoantibodies in Hypersensitivity Pneumonitis

A major genetic determinant of autoimmune diseases is associated with the presence of autoantibodies in Hypersensitivity Pneumonitis

Hypersensitivity pneumonitis (HP) is an immune-mediated illness triggered by publicity to natural particles in prone people. It has been reported {that a} subgroup of sufferers with HP develops autoantibodies with or with out scientific manifestations of autoimmune illness. However, the mechanisms concerned in this course of and the impact of the autoantibodies on scientific course in HP is unknown. We evaluated the affiliation between HLA class II alleles and HP sufferers with and with out autoantibodies.

METHODS

One hundred seventy HP sufferers had been included. We analysed the presence of antinuclear antibodies, rheumatoid issue, anti-SSA/Ro, anti-SSB/La, and anti-CCP at the time of prognosis. In addition, in a subset of sufferers, we evaluated anti-Scl-70, ANCA, and anti-DNA. HLA typing was carried out by PCR-SSP in a high-resolution modality, together with HLA-DRB1 and HLA-DQB1 loci. Statistical evaluation was carried out using Epi-Info v7 and SPSSv20.

RESULTS

Sixty HP sufferers confirmed sera autoantibodies (HPAbs+), and 110 HP sufferers didn’t (HPAbs-). The frequency of the allele HLA-DRB1*03:01 was remarkably elevated in the HPAbs+ group (10.8% versus 0.45%; OR=30.14, 95%CI 3.83-237.1; p=1.65E-04 after Bonferroni’s correction). Likewise, we discovered that the haplotype DRB1*03:01-DQB1*02:01, which is half of the 8.1 ancestral haplotype, a major genetic determinant of autoimmune diseases confers vital danger to develop autoantibodies (OR=19.23, 95%CI 2.37-155.9; p=0.0088 after Bonferroni’s correction).

A major genetic determinant of autoimmune diseases is associated with the presence of autoantibodies in Hypersensitivity Pneumonitis
A major genetic determinant of autoimmune diseases is associated with the presence of autoantibodies in Hypersensitivity Pneumonitis

Also, the HLA-DRB1*03:01 allele was associated with increased mortality in sufferers with HP (adjusted OR=5.9, 95%IC 1.05-33.05; p=0.043).A subset of HP sufferers presents circulating autoantibodies and better mortality, which are associated with some alleles of 8.1 ancestral haplotype.

ggroups: an R package deal for pedigree and genetic teams information

R is a multi-platform statistical software program and an object oriented programming language. The package deal archive community for R offers CRAN repository that options over 15,000 free open supply packages, at the time of writing this text (https://cran.r-project.org/web/packages, accessed in October 2019). The package deal ggroups is launched in this text.

The goal of this package deal is offering features for checking and processing the pedigree, calculation of the additive genetic relationship matrix and its inverse, that are used to review the inhabitants construction and predicting the genetic benefit of animals. Calculation of the dominance relationship matrix and its inverse are additionally coated.

A idea in animal breeding is genetic teams, which is about the inequality of the common genetic deserves for teams of unknown mother and father. The package deal offers features for the calculation of the matrix of genetic group contributions (Q). Calculating Q is computationally demanding, and relying on the dimension of the pedigree and the quantity of genetic teams, it may not be possible utilizing private computer systems.

Therefore, a computationally optimised perform and its parallel processing different are offered in the package deal.Using pattern information, outputs from completely different features of the package deal had been introduced as an example an actual expertise of working with the package deal.

The introduced R package deal is a free and open supply software primarily for quantitative geneticists and ecologists, who deal with pedigree information. It offers quite a few features for dealing with pedigree information, and calculating varied pedigree-based matrices. Some of the features are computationally optimised for large-scale information.

To consider the scientific significance of the information obtained throughout enzyme-linked immunosorbent assay (ELISA) of the blood serum of sufferers with inflammatory diseases of the anterior eye section in comparison with a gaggle of wholesome volunteers.

MATERIAL AND METHODS

A retrospective evaluation of the outcomes of serum ELISA of 200 sufferers with persistent keratoconjunctivitis and keratouveitis was carried out utilizing the solid-phase IFA methodology in order to detect the presence of antibodies to herpes simplex viruses of 1 and a pair of sorts (HSV 1, 2), cytomegalovirus (CMV), Chlamydia trachomatis and Toxoplasma gondii. The management group consisted of 34 wholesome volunteers with no indicators of irritation of the eye tissue.

RESULTS

There had been no vital variations in the frequency of detection of each «acute-phase» IgM and «persistent» IgG to HSV and CMV between sufferers of the major group and wholesome volunteers (ρ≥0.05). The ranges of IgG to Toxoplasma gondii in the examine group had been 3 times increased than in the management group (ρ≤0.05). A comparative evaluation of the frequency of detection of antibodies to Chlamydia trachomatis confirmed statistically vital variations between the teams and extra frequent detection of immunoglobulins courses A, G and M in sufferers with inflammatory eye diseases (ρ≤0.05).

The absence of statistical variations with the management group in the frequency of detection of lively HSV and CMV infections in sufferers with inflammatory diseases of the anterior eye section when utilizing ELISA permits to advocate the use of further strategies of examination akin to polymerase chain response (PCR) and real-time PCR (RT-PCR) with identification of pathogen genetic materials in the obtainable organic secretions.

The enhance in IgG titers to toxoplasma in sufferers of the major group apparently signifies a cross-stimulation of antibody synthesis in opposition to the background of a persistent inflammatory course of.

Significant variations in the frequency of detection of all kinds of antibodies to chlamydiae suggests a major etiological position of this pathogen in the growth and upkeep of persistent irritation in the anterior eye section.

Effects of CYP3A5 Polymorphisms on Efficacy and Safety of Tacrolimus Therapy in Patients with Idiopathic Membranous Nephropathy

Effects of CYP3A5 Polymorphisms on Efficacy and Safety of Tacrolimus Therapy in Patients with Idiopathic Membranous Nephropathy

Tacrolimus (TAC) is helpful for sufferers with idiopathic membranous nephropathy (IMN). It has a slim therapeutic focus vary and many components affect TAC blood focus.

CYP3A5 is an important enzyme in TAC metabolism. The intention of this examine was to investigate the consequences of CYP3A5 gene polymorphisms on the efficacy and security of TAC in IMN sufferers.Patients with IMN who acquired oral TAC (0.05-0.075mg/kg/day) mixed with prednisone (0.5mg/kg/day) from March 2016 to October 2018 had been included. The information of medical traits, therapeutic medication and opposed reactions of sufferers had been collected at baseline and throughout 24 weeks of therapy. Patients had been divided into two teams in accordance with completely different CYP3A5 genetic polymorphisms. The important variations in the efficacy and uncomfortable side effects between the 2 teams had been analyzed.

Results

A complete of 76 sufferers who accomplished follow-up had been divided into CYP3A5 nonexpresser (CYP3A5*3/*3) group and CYP3A5 expresser (CYP3A5 *1/*3) group. The important affiliation between the CYP3A5 phenotype and TAC metabolism was noticed. A complete of 43 case-times sufferers exhibited opposed results. The an infection fee in CYP3A5 nonexpresser group (21.95%) was remarkably larger than the speed in CYP3A5 expresser group (5.71%). Blood focus and C0/D ranges had been threat components for opposed occasions via logistic regression evaluation.

There was no statistical distinction between the examine teams with respect to the efficacy.Our outcomes demonstrated that CYP3A5 polymorphisms had essential guiding roles in the therapy of IMN with tacrolimus. CYP3A5 expressers required larger every day doses of TAC to realize the goal drug focus, however with fewer uncomfortable side effects. CYP3A5 genetic polymorphism is perhaps used for TAC dosing adjustment to optimize the therapy for sufferers with IMN.

TS: a strong truncated take a look at to detect novel illness related genes utilizing publicly obtainable gWAS abstract information

In the final decade, a big quantity of frequent variants underlying advanced illnesses have been recognized via genome-wide affiliation research (GWASs). Summary information of the GWASs are freely and publicly obtainable. The abstract information is normally obtained via single marker evaluation.

Effects of CYP3A5 Polymorphisms on Efficacy and Safety of Tacrolimus Therapy in Patients with Idiopathic Membranous Nephropathy
Effects of CYP3A5 Polymorphisms on Efficacy and Safety of Tacrolimus Therapy in Patients with Idiopathic Membranous Nephropathy

Gene-based evaluation presents a helpful various and complement to single marker evaluation. Results from gene stage affiliation exams could be extra readily built-in with downstream purposeful and pathogenic investigations. Most present gene-based strategies fall into two classes: burden exams and quadratic exams. Burden exams are normally highly effective when the instructions of results of causal variants are the identical.

However, they might endure loss of statistical energy when completely different instructions of results exist on the causal variants. The energy of quadratic exams just isn’t affected by the instructions of results however may very well be much less highly effective attributable to points corresponding to the big quantity of diploma of freedoms.

These drawbacks of present gene primarily based strategies motivated us to develop a brand new highly effective methodology to determine illness related genes utilizing present GWAS abstract information.In this paper, we suggest a brand new truncated statistic methodology (TS) by using a truncated methodology to search out the genes which have a real contribution to the genetic affiliation.

Extensive simulation research show that our proposed take a look at outperforms different comparable exams. We utilized TS and different comparable strategies to the schizophrenia GWAS information and sort 2 diabetes (T2D) GWAS meta-analysis abstract information. TS recognized extra illness related genes than comparable strategies.

Many of the numerous genes recognized by TS could have essential mechanisms related to the related traits. TS is carried out in C program TS, which is freely and publicly obtainable on-line.The proposed truncated statistic outperforms present strategies. It could be employed to detect novel traits related genes utilizing GWAS abstract information.

A User-Friendly, Web-Based Integrative Tool (ESurv) for Survival Analysis: Development and Validation Study

A User-Friendly, Web-Based Integrative Tool (ESurv) for Survival Analysis: Development and Validation Study
A User-Friendly, Web-Based Integrative Tool (ESurv) for Survival Analysis: Development and Validation Study
A User-Friendly, Web-Based Integrative Tool (ESurv) for Survival Analysis: Development and Validation Study

Prognostic genes or gene signatures have been extensively used to foretell affected person survival and assist in making choices pertaining to therapeutic actions. Although some web-based survival evaluation instruments have been developed, they’ve a number of limitations.

Taking these limitations into consideration, we developed ESurv (Easy, Effective, and Excellent Survival evaluation instrument), a web-based instrument that may carry out superior survival analyses utilizing user-derived knowledge or knowledge from The Cancer Genome Atlas (TCGA).

Users can conduct univariate analyses and grouped variable alternatives utilizing multiomics knowledge from TCGA.We used R to code survival analyses primarily based on multiomics knowledge from TCGA. To carry out these analyses, we excluded sufferers and genes that had inadequate data. Clinical variables had been labeled as 0 and 1 when there have been two classes (for instance, chemotherapy: no or sure), and dummy variables had been used the place options had three or extra outcomes (for instance, with respect to laterality: proper, left, or bilateral).

Through univariate analyses, ESurv can determine the prognostic significance for single genes utilizing the survival curve (median or optimum cutoff), space below the curve (AUC) with C statistics, and receiver working traits (ROC).

Users can acquire prognostic variable signatures primarily based on multiomics knowledge from scientific variables or grouped variable alternatives (lasso, elastic web regularization, and network-regularized high-dimensional Cox-regression) and choose the identical outputs as above.

In addition, customers can create customized gene signatures for particular cancers utilizing numerous genes of curiosity. One of a very powerful features of ESurv is that customers can carry out all survival analyses utilizing their very own knowledge.

Using superior statistical methods appropriate for high-dimensional knowledge, together with genetic knowledge, and built-in survival evaluation, ESurv overcomes the restrictions of earlier web-based instruments and will assist biomedical researchers simply carry out advanced survival analyses.

Diversity of navel microbiome in younger adults

Introduction. Human pores and skin microbial communities characterize an incredible supply of genetic variety that evolves as a perform of human age. Microbiota differs between areas of oily and moist pores and skin, and seems to stabilize with age.

Aim. We have a minimal understanding of the time-frame required for the stabilization of pores and skin microbiota, and the function performed by gender. In the present research, we examined the microbiota current within the navel area of college-attending younger adults within the age group of 18-25 years and investigated if variety is related to gender (male and feminine).

Method. The research concerned 16 feminine and six male topics. Isolated DNA samples from navel swabs had been processed utilizing the Nextera XT library preparation equipment and sequenced utilizing the MiSeq platform. Data had been analysed utilizing QIIME and statistical evaluation carried out in R.

Results. Microbiota of navel pores and skin is dominated by Corynebacterium and Staphylococcus and contains opportunistic pathogens like Clostridium and Pseudomonas.

Also current as the most important part of the flora had been the organisms usually related to the gastrointestinal tract akin to AcinetobacterCampylobacterKlebsiella and organisms from the Enterobacteriaceae and Moraxellaceae households. Comparison of alpha and beta variety of the microbiota within the male and feminine navel areas means that the flora shouldn’t be statistically totally different (P>0.05). However, pairwise comparability means that the abundance of 12 particular genera various with gender, together with larger abundance of Klebsiella and Enterobacter in females.

Conclusion. Our findings point out that the navel pores and skin microbiota of younger adults has a core microbiota of Corynebacterium and Staphylococcus. We additionally famous the presence of a big variety of opportunistic pathogens. A minor gender distinction within the abundance of particular person organisms was additionally noticed.

What is DNA?

DNA

DNA, DeoxyriboNucleic Acid is a long polymer that carries the heriditary informantion of most of organisms, DNA is found in the nuclei of eukaryote cells and in the nucleiod of bacteria, we found also the DNA in some organells ( mitochondria and cell plastids (chloroplasts)) and in viruses.

Structure of DNA

The main structure of DNA is double helix which is composed of two antiparallel strands, these strands are made up of the sugar 2-deoxyribose, phosphate, and purine and pyrimidine bases.

 Each phosphate is linked to the 3′ position of the preceding deoxyribose and to the 5′ position of the following deoxyribose.

There are four bases found in the DNA:

Adenine (A)

Tthymine (T)

Guanine (G)

Cytosine (C)

Adenine, A, is always paired with thymine, T.

Cytosine, C, is always paired with guanine, G.

Each 2-deoxyribose is linked to one of four bases in the 1′ position via a covalent glycosidic bond.

we call a nucleoside a base linked to a sugar

we call a nucleotide a base linked to a sugar and a phosphate

Function

DNA play a great role in:

  • Cell living and growing
  • Maintain and propagation of genetic information from generation to generation
  • Synthesis the components of the cells

Replication of DNA

Each strand of the DNA contains all the information needed to form a new double stranded molecules.

after separtion of the double helix each strand is used as a template to form the new strand of DNA, this is carried out by enzymes called DNA polymerases, All DNA polymerases synthesize new DNA in a 5′ to 3′ direction.

Transcription

Transcription is the processus of recopyin genetic information in DNA to form RNA (RiboNucleic Acid) by using RNA polymerases, the result RNA is used to make proteins by the cellular protein-synthesizing (translation) machinery.

After the synthesis of RNA, the DNA reverts to its double helix form.

Genetics

Genetics

What is Genetics

Genetic is the science that studies the behavior and nature of genes, a gene is a basic unit of heredity, so the genetics studies the variation and heredity in living organisms.

Short history of Genetics

  • Gregor Mendel is the father of Genetics, in 1865, After many experiences on hybridizing pea plants he reported that many series of traits passed down through generations.
  • In 1866, Mendel noted certain patterns of inheritance seemed to be transmitted independently of one another.
  • It wasn’t until 1900 that the scientific community recognize the observation of Mendel, and thus when three Europeans botanists reported the result of their breeding experiments and liked it to Mendel’s work.
  • The biologist at the university of Cambridge, William Bateson was exited to study the relation between Mendel’s observations and evolution, and he recruited for that a number of young scientist women. Later they noted that Mendel’s principles extended across the plant and animals.
  • In 2001, Willian Bateson named this discipline “Genetics”, and “Gene” the unit of this discipline is the factor that was inherited.

Study

The Genetics studies the difference between the individuals. If there is a difference it is transmitted to the next generation by sexual reproduction, or other process. In brief genetics studies the descendance across diverse individuals through at least two successive generations.

The mechanism of heredity depends on probabilities, but only some statistical concepts are needed.

Molecular Genetics

The research of Morgan on the fly proved that the genes were on the chromosomes.

But it was still unknown what genes were made up of, what was their molecular structure, and biologists debates the genetic material was DNA or proteins.

In 1952, Alfred Hershey and Martha Chase at Cold Spring Harbor Laboratory ingeniously used viruses (proteins and DNA inside) and proved by a radioactive experience that the genetic material is the DNA.

After many research, James Watson and Francis Crick at the University of Cambridge determined that DNA was a double helix, and the structure is two strands wrapped around each other.

DNA (DeoxyriboNucleic Acid) is a very long polymer made up of four different nucleotides (Adenine (A), Guanine (G), Cytosine (C), and Thymine (T).). A always bind to T, and G always to C.

These long double helix strands make up chromosomes, chromosomes divided into thousand of unit to form genes.

Molecular biologists spent the 1950s and 1960s using the double helix structure of DNA elucidate the mechanism of genetic replication and function.

Gene Role in Organisms

  • DNA is made into RNA
  • RNA specifies the structure of a specific protein
  • DNA is transcribed into mRNA (RNA messenger) by the enzyme RNA polymerase (specific DNA sequences ensure that RNA polymerase transcribes certain genes only in the correct cell type at the proper time.)
  • The mRNA serves for the protein synthesis by using the ribosomes and tRNAs.

Role of Molecular Genetic

  • Learning what is the role of DNA in determining the regulation of gene expression.
  • DNA replication in cells.
  • Determination of the complete DNA Sequence of several organisms.
  • Determination of the sequence of proteins.
  • Trying to understand how the sequence of a proteins determines its function.
  • Study of mutations that results of the errors during the replication of DNA.