Phylogenetic tree inferred by using Maximum Likelihood (ML) and Bayesian inference (BI).

<p>TPM3+I+G was the best fitting mutation model. For ML 1000 bootstrap replicates were considered and for BI posterior probabilities were calculated; posterior probabilities (<b>bold</b>) over 0.95 and bootstrap confidence (<i>italic</i>) over 90% are considered valid support and are shown in the tree.</p

Evolution of C, D and S-Type Cystatins in Mammals: An Extensive Gene Duplication in Primates

<div><p>Cystatins are a family of inhibitors of cysteine peptidases that comprises the salivary cystatins (D and S-type cystatins) and cystatin C. These cystatins are encoded by a multigene family (<i>CST3</i>, <i>CST5</i>, <i>CST4</i>, <i>CST1</i> and <i>CST2</i>) organized in tandem in the human genome. Their presence and functional importance in human saliva has been reported, however the distribution of these proteins in other mammals is still unclear. Here, we performed a proteomic analysis of the saliva of several mammals and studied the evolution of this multigene family. The proteomic analysis detected S-type cystatins (S, SA, and SN) in human saliva and cystatin D in rat saliva. The evolutionary analysis showed that the cystatin C encoding gene is present in species of the most representative mammalian groups, i.e. Artiodactyla, Rodentia, Lagomorpha, Carnivora and Primates. On the other hand, D and S-type cystatins are mainly retrieved from Primates, and especially the evolution of S-type cystatins seems to be a dynamic process as seen in <i>Pongo abelii</i> genome where several copies of <i>CST1-like</i> gene (cystatin SN) were found. In Rodents, a group of cystatins previously identified as D and S has also evolved. Despite the high divergence of the amino acid sequence, their position in the phylogenetic tree and their genome organization suggests a common origin with those of the Primates. These results suggest that the D and S type cystatins have emerged before the mammalian radiation and were retained only in Primates and Rodents. Although the mechanisms driving the evolution of cystatins are unknown, it seems to be a dynamic process with several gene duplications evolving according to the birth-and-death model of evolution. The factors that led to the appearance of a group of saliva-specific cystatins in Primates and its rapid evolution remain undetermined, but may be associated with an adaptive advantage.</p></div

Type-II cystatin gene cluster region overview.

<p>Organization of the <i>CST1</i>-<i>5</i> genes in human (<i>Homo sapiens</i>), chimpanzee (<i>Pan troglodytes</i>), orangutan (<i>Pongo abelii</i>), gorilla (<i>Gorilla gorilla</i>), rhesus monkey (<i>Macaca mulatta</i>), marmoset (<i>Callithrix jacchus</i>), rat (<i>Rattus norvegicus</i>), mouse (<i>Mus musculus</i>), pig (<i>Sus scrofa</i>), cattle (<i>Bos taurus</i>) and dog (<i>Canis lupus familiaris</i>). The transcriptional orientation of the genes is shown; the pseudogenes are highlighted in yellow (data from ENSEMBL and NCBI databases). The genes used in the subsequent analysis are highlighted in blue.</p

Amino acid composition of cystatins.

<p><i>Homo sapiens</i> cystatin C (P01034); <i>Bos taurus</i> cystatin C (P01035); <i>Rattus norvegicus</i> cystatin C (P14841); <i>Canis lupus familiaris</i> cystatin C (J9NS29); <i>Homo sapiens</i> cystatin D (P28325); <i>Callithrix jacchus</i> cystatin D (ENSCJAP00000001156); <i>Macaca mulatta</i> cystatin D (G7N352); <i>Homo sapiens</i> cystatin SN (P01037); <i>Pan troglodytes</i> cystatin SN (H2QK35); <i>Homo sapiens</i> cystatin S (P01036); <i>Pan troglodytes</i> cystatin S (H2QK34); <i>Homo sapiens</i> cystatin SA (P09228); <i>Pan troglodytes</i> cystatin SA (H2QK36) and <i>Rattus norvegicus</i> cystatin S (P19313). Filled grey boxes indicate conserved amino acid motifs; empty boxes indicate conserved amino acids characteristic of each cystatin; asterisks (*) mark the codons on <i>CST3</i> under negative selection.</p

Cystatins found by LC-MS/MS from human, dog, sheep, cattle, horse, rabbit and rat saliva.

<p>Cystatins found by LC-MS/MS from human, dog, sheep, cattle, horse, rabbit and rat saliva.</p

Diagram of salivary cystatin evolution.

<p>Cystatin genes estimated emergence is shown (divergence times were based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109050#pone.0109050-Hedges1" target="_blank">[45]</a>).</p

Transducer positioning and resulting ultrasound image obtained in a dorsal approach in the dorsal plane of the right hip joint.

A: Rabbit placement in lateral recumbency with the probe at the level of the joint, and corresponding anatomic specimen. The red rectangle and asterisk represent the probe orientation and indicator, respectively. B: Sonoanatomy of the hip: (1) skin and subcutaneous tissue, (2) fascia glutea, (3) gluteofemoralis muscle, (4) gluteus superficialis muscle, (5) gluteus accessorius muscle, (6) gluteus medius muscle, (7) gluteus profundus muscle, (8) articular cartilage of the femoral head, and (9) femoral head. Do: Dorsal, Cr: cranial, and Ca: caudal.</p

Summary of the Dunn’s Multiple Comparison test in the image quality assessment for each plane in 11 rabbit cadavers (n = 22 hips), not considering each articular or periarticular structure individually but as a collective unit.

Summary of the Dunn’s Multiple Comparison test in the image quality assessment for each plane in 11 rabbit cadavers (n = 22 hips), not considering each articular or periarticular structure individually but as a collective unit.</p

Transducer positioning and resulting ultrasound image obtained in a ventral approach in the sagittal plane of the left hip joint.

A: Rabbit placement in dorsal recumbency with the probe at the level of the joint, and corresponding anatomic specimen. The red rectangle and asterisk represent the probe orientation and indicator, respectively. B: Sonoanatomy of the hip: (1) skin and subcutaneous tissue, (2) sartorius muscle, (3) vastus medialis muscle, (4) rectus femoris muscle, (5) articular cartilage of the femoral head, and (6) femoral head. Ve: ventral, Cr: cranial, and Ca: caudal.</p