A new physical map of the bovine genome has been constructed by integrating data from genetic and radiation hybrid maps, and a new bovine BAC map, with the bovine genome draft assembly.
The domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction.
Here we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found.
The map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.
The availability of a high-density SNP genotyping chip and a reference genome sequence of the pig (Sus scrofa) enabled the construction of a high-density linkage map. A high-density linkage map is an essential tool for further fine-mapping of quantitative trait loci (QTL) for a variety of traits in the pig and for a better understanding of mechanisms underlying genome evolution.
Four different pig pedigrees were genotyped using the Illumina PorcineSNP60 BeadChip. Recombination maps for the autosomes were computed for each individual pedigree using a common set of markers. The resulting genetic maps comprised 38,599 SNPs, including 928 SNPs not positioned on a chromosome in the current assembly of the pig genome (build 10.2). The total genetic length varied according to the pedigree, from 1797 to 2149 cM. Female maps were longer than male maps, with a notable exception for SSC1 where male maps are characterized by a higher recombination rate than females in the region between 91–250 Mb. The recombination rates varied among chromosomes and along individual chromosomes, regions with high recombination rates tending to cluster close to the chromosome ends, irrespective of the position of the centromere. Correlations between main sequence features and recombination rates were investigated and significant correlations were obtained for all the studied motifs. Regions characterized by high recombination rates were enriched for specific GC-rich sequence motifs as compared to low recombinant regions. These correlations were higher in females than in males, and females were found to be more recombinant than males at regions where the GC content was greater than 0.4.
The analysis of the recombination rate along the pig genome highlighted that the regions exhibiting higher levels of recombination tend to cluster around the ends of the chromosomes irrespective of the location of the centromere. Major sex-differences in recombination were observed: females had a higher recombination rate within GC-rich regions and exhibited a stronger correlation between recombination rates and specific sequence features.
Pig; Recombination; Genome; SNP; Linkage; Meiosis; Telomere; Centromere; Isochore
The pig, Sus scrofa domestica includes both the miniature and commercial domestic breed. These animals have influenced the human life and economies and have been studied throughout history. Although the miniature breeds are more recent and have increasingly been used in a variety of biomedical studies, their cell lines have rarely been established. Therefore, we sought to establish primary and immortal cell lines derived from both the miniature and domestic pig to better enable insight into possible in vivo growth differences.
The in vitro lifespan of primary domestic pig fibroblast (PF) and miniature pig fibroblast (MPF) cells using a standard 3T3 protocol was determined. Both of the primary PF and MPF cells were shown to have a two-step replicative senescence barrier. Primary MPF cells exhibited a relatively shorter lifespan and slower proliferation rate compared to those of primary PF cells. Beyond senescence barriers, lifespan-extended PF and MPF cells were eventually established and indicated spontaneous cellular immortalization. In contrast to the immortalized PF cells, immortal MPF cells showed a transformed phenotype and possessed more frequent chromosomal abnormalities and loss of p53 regulatory function. The lifespan of primary MPF and PF cells was extended by inactivation of the p53 function using transduction by SV40LT without any detectable senescent phenotype.
These results suggest that p53 signaling might be a major determinant for the replicative senescence in the MPF cells that have the shorter lifespan and slower growth rate compared to PF cells in vitro.
The aim of this project was to develop a methodology to introduce wireless video capsule endoscopy in preclinical research. Five mature female pigs (Sus scrofa domestica) were selected for the study. Capsule endoscopes (the EndoCapsule system; Olympus) were introduced into the duodenum endoscopically in each of the animals. The life span of batteries (i.e., total time of endoscopy recording) was 487–540 min (median 492 min). The capsule endoscope reached the cecum during enteroscopy once (after 7 h 57 min), in the remaining cases, endoscopy recordings terminated in the distal or terminal ileum. All capsule enteroscopies found a normal pattern of the small intestine. The intestinal lumen is narrower, transverse folds are sparse or even absent, villi are wider but less prominent in pigs compared to humans. Capsule endoscopy in experimental pigs will be helpful for future trials on injury of different drugs and xenobiotics to the small bowel.
Capsule endoscopy; Enteroscopy; Experimental pigs; Small bowel
The lack of a Near Eastern genetic signature in modern European porcine breeds indicates that, although domestic pigs from the Fertile Crescent entered Europe during the Neolithic, they were completely replaced by their European counterparts in a short window of time. Whilst the absence of such genetic signature has been convincingly demonstrated at the mitochondrial level, variation at the autosomal genomes of European and Near Eastern Sus scrofa has not been compared yet. Herewith, we have explored the genetic relationships among 43 wild boar from Europe (N = 21), Near East (N = 19) and Korea (N = 3), and 40 Iberian (N = 16), Canarian (N = 4) and Mangalitza (N = 20) pigs by using a high throughput SNP genotyping platform. After data filtering, 37,167 autosomal SNPs were used to perform population genetics analyses. A multidimensional scaling plot based on genome-wide identity-by-state pairwise distances inferred with PLINK showed that Near Eastern and European wild boar populations are genetically differentiated. Maximum likelihood trees built with TreeMix supported this conclusion i.e. an early population split between Near Eastern and European Sus scrofa was observed. Moreover, analysis of the data with Structure evidenced that the sampled Iberian, Canarian and Mangalitza pigs did not carry any autosomal signature compatible with a Near Eastern ancestry, a finding that agrees well with previous mitochondrial studies.
Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.
The centromere is a strange locus that derives its identity from the proteins that shape it rather than the DNA sequences it contains. It also functions in a remarkably singular way, providing a motor and command control center for the chromosome in conjunction with the kinetochore. Key to centromere identity is the chromatin that comprises it, which has a unique nucleosomal “bead on a string” including a special centromeric histone H3, called CENP-A. Found in alternating clusters of nucleosomes with “regular” histone H3, CENP-A is crucial for propagating centromere identity as well as for regulating kinetochore function. In this study, we have analysed the cell cycle dynamics of CENP-T and CENP-W, another two components of the constitutive centromere associated network. We show that, unlike CENP-A, CENP-T/W are not inherited stringently by daughter cells. Instead, these complexes - which are bound to the interstitial “regular” H3 nucleosome domains - assemble after DNA replication and are required for kinetochore formation. Thus, we propose that a stable CENP-A nucleosome population plays a role in centromere locus inheritance to daughter cells, while dynamic CENP-T/W and H3 nucleosomes provide a cycling function that triggers kinetochore assembly as cells enter mitosis in each new cell cycle.
Mycobacterium celatum, a slowly growing potentially pathogenic mycobacterium first described in humans, is regarded as an uncommon cause of human infection, though capable of inducing invasive disease in immunocompromised hosts. According to some reports, a serious disease due to M. celatum may also occur in individuals with no apparent immunodeficiency. In animals, an M. celatum-related disease has been described in three cases only: twice in a domestic ferret (Mustela putorius furo) and once in a white-tailed trogon (Trogon viridis). In this paper, we report the first detection of M. celatum in a domestic pig (Sus scrofa domestica) and roe deer (Capreolus capreolus). A nation-wide overview of human M. celatum infections recorded in Slovenia between 2000 and 2010 is also given. Pulmonary disease due to M. celatum was recognized in one patient with a history of a preexisting lung disease.
Erysipelas is an animal disease caused by Gram-positive bacteria Erysipelothrix rhusiopathiae. Among the domestic animals, domestic pig (Sus scrofa f. domestica) suffers most frequently from the disease in human environment. This is a typical animal-borne disease observed mainly in occupational groups employed in agriculture, farming (of animals and birds), fishing and manufacturing industry.
We are presenting the clinical course of infection (E. rhusiopathiae) and discuss clinical forms. E. rhusiopathiae in humans may have the following clinical course: mild form of skin infection diagnosed as local erythema (erysipeloid), disseminated form of skin infection and the most serious form of infection of systemic course (endocarditis and sepsis). Mild skin infection and local erythema are the most common forms. Very rare case of animal-borne infection course has been presented in which after initial phase the disease was generalised to the abscesses formation in paravertebral space, spondylitis and empyema formation in spinal canal. In the presented clinical case, the patient was suffering from diabetes. It was probably an additional risk factor of the disease generalisation. Patient underwent drainage of empyema in spinal canal, after which his neurological status gradually improved. Antibiotic therapy was implemented and continued for 8 weeks. Such course of erysipelas was not previously described in the literature.
After therapy neurological status was improved. In follow MRI control exam empyema and spondylitis was successfully eliminated.
Various complications of the disease, such as endocarditis and heart valves disturbances, are well known and are the most severe complications of the generalised infection. Proper targeted and long-term antibiotic therapy is crucial.
Zoonosis; Erysipelothrix rhusiopathiae; Abscesses in paravertebral space; Spondylitis
Studying vascular anatomy, especially in the context of relationships with hard tissues, is of great interest to biologists. Vascular studies have provided significant insight into physiology, function, phylogenetic relationships, and evolutionary patterns. Injection of resin or latex into the vascular system has been a standard technique for decades. There has been a recent surge in popularity of more modern methods, especially radiopaque latex vascular injection followed by CT scanning and digital “dissection.” This technique best displays both blood vessels and bone, and allows injections to be performed on cadaveric specimens. Vascular injection is risky, however, because it is not a standardizable technique, as each specimen is variable with regard to injection pressure and timing. Moreover, it is not possible to view the perfusion of injection medium throughout the vascular system of interest. Both data and rare specimens can therefore be lost due to poor or excessive perfusion. Here, we use biplanar video fluoroscopy as a technique to guide craniovascular radiopaque latex injection. Cadaveric domestic pigs (Sus scrofa domestica) and white-tailed deer (Odocoileus virginianus) were injected with radiopaque latex under guidance of fluoroscopy. This method was found to enable adjustments, in real-time, to the rate, location, and pressure at which latex is injected in order to avoid data and specimen loss. In addition to visualizing the injection process, this technique can be used to determine flow patterns, and has facilitated the development of consistent markers for complete perfusion.
Mild therapeutic hypothermia (HT) has been implemented in the management of post cardiac arrest (CA) syndrome after the publication of clinical trials comparing HT with common practice (ie, usually hyperthermia). Current evidence on the comparison between therapeutic HT and controlled normothermia (NT) in CA survivors, however, remains insufficient.
Eight female swine (sus scrofa domestica; body weight 45 kg) were randomly assigned to receive either mild therapeutic HT or controlled NT, with four animals per group. Veno-arterial extracorporeal membrane oxygenation (ECMO) was established and at minimal ECMO flow (0.5 L/min) ventricular fibrillation was induced by rapid ventricular pacing. After 20 min of CA, circulation was restored by increasing the ECMO flow to 4.5 L/min; 90 min of reperfusion followed. Target core temperatures (HT: 33°C; NT: 36.8°C) were maintained using the heat exchanger on the oxygenator. Invasive blood pressure was measured in the aortic arch, and cerebral oxygenation was assessed using near-infrared spectroscopy. After 60 min of reperfusion, up to three defibrillation attempts were performed. After 90 min of reperfusion, blood samples were drawn for the measurement of troponin I (TnI), myoglobin (MGB), creatine-phosphokinase (CPK), alanin-aminotransferase (ALT), neuron-specific enolase (NSE) and cystatin C (CysC) levels. Reactive oxygen metabolite (ROM) levels and biological antioxidant potential (BAP) were also measured.
Significantly higher blood pressure and cerebral oxygenation values were observed in the HT group (P<0.05). Sinus rhythm was restored in all of the HT animals and in one from the NT group. The levels of TnI, MGB, CPK, ALT, and ROM were significantly lower in the HT group (P<0.05); levels of NSE, CysC, and BAP were comparable in both groups.
Our results from animal model of cardiac arrest indicate that HT may be superior to NT for the maintenance of blood pressure, cerebral oxygenation, organ protection and oxidative stress suppression following CA.
Cardiac arrest; Mild hypothermia; Normothermia; Post-cardiac arrest syndrome; Extracorporeal membrane oxygenation; Cerebral oxygenation; Blood pressure; Organ protection; Oxidative stress
The distribution of the points of breakage and reunion of a series of 58 Robertsonian translocations, 53 reciprocal translocations, and 10 inversions is described. An excess of 13/14 and 14/21 rearrangements was found among the Robertsonian translocations, this excess being independent of the method of ascertainment of the proband. The distribution of break points between chromosome arms in the reciprocal translocations, with the possible exception of the long arms of chromosome 11, was no different from that expected on the basis of their relative lengths. However, within arms there appeared to be an excess of breaks in the terminal regions, an excess of terminal/centromeric translocations where ascertainment was through a balanced carrier and a possible excess of terminal/median translocations where ascertainment was through an unbalanced carrier. Nine inversions were analysed and three of these involved identical break points on chromosome 8.
Possible reasons for the apparent non-randomness of points of breakage and exchange are discussed and it is concluded that the techniques of preparation, methods of observations, and methods of ascertainment all affect the distribution of observed points of breakage and exchange and must therefore be taken into cognizance in any study of chromosome rearrangements in man.
Indirect Genetic Effects (IGEs), also known as associative effects, are the heritable effects that an individual has on the phenotype of its social partners. Selection for IGEs has been proposed as a method to reduce harmful behaviours, in particular aggression, in livestock and aquaculture. The mechanisms behind IGEs, however, have rarely been studied. The objective was therefore to assess aggression in pigs which were divergently selected for IGEs on growth (IGEg). In a one generation selection experiment, we studied 480 offspring of pigs (Sus scrofa) that were selected for relatively high or low IGEg and housed in homogeneous IGEg groups in either barren or enriched environments. Skin lesion scores, a proxy measure of aggression, and aggressive behaviours were recorded. The two distinct IGEg groups did not differ in number of skin lesions, or in amount of reciprocal fighting, both under stable social conditions and in confrontation with unfamiliar pigs in a 24 h regrouping test. Pigs selected for a positive effect on the growth of their group members, however, performed less non-reciprocal biting and showed considerably less aggression at reunion with familiar group members after they had been separated during a 24 h regrouping test. The enriched environment was associated with more skin lesions but less non-reciprocal biting under stable social conditions. Changes in aggression between pigs selected for IGEg were not influenced by G×E interactions with regard to the level of environmental enrichment. It is likely that selection on IGEg targets a behavioural strategy, rather than a single behavioural trait such as aggressiveness.
To report the postoperative courses of 2 patients with Fuchs heterochromic iridocyclitis (FHI) and the concentrations of various cytokines, chemokines and growth factors in vitreous fluid samples to obtain insights into pathobiochemical aspects. Subjects: The patients were a 27- and a 47-year-old woman. Phacoemulsification and aspiration, intraocular lens (IOL) implantation, and pars plana vitrectomy were performed to treat their cataracts and vitreous opacities. During their early postoperative periods, inflammatory cells precipitated on the IOL and intraocular pressure was increased in both patients.
At the time of surgery, undiluted vitreous fluid specimens were collected. The concentrations of multiple cytokines, chemokines and growth factors were measured by a bead array immunodetection system.
The levels of interleukin-1ra, −5, −6, −8, −10 and −13, interferon-inducible 10-kDa protein, monocyte chemoattractant protein 1, macrophage inflammatory protein 1β, and regulated upon activation, normal T-cell expressed and secreted (RANTES) were significantly elevated in vitreous fluid in both patients.
Although the postoperative course was generally favorable in patients with FHI, steroid instillation was necessary for a few months postoperatively, as precipitates easily formed on the IOL surface and elevated intraocular pressure. The profiles of intravitreal concentrations of cytokines, chemokines and growth factors may characterize postoperative inflammatory reactions.
Fuchs heterochromic iridocyclitis; Cytokines; Chemokines; Growth factors; Interleukins
We are reporting a case of bilateral Fuchs’ heterochromic iridocyclitis with chikungunya virus infection in the left eye. A 20-year-old female was presented with a past history of fever suggestive of chikungunya with bilateral Fuchs’ heterochromic iridocyclitis and complicated cataract. She had a tripod dendritic pattern of keratic precipitates by confocal microscopy in the left eye with a stippled pattern of keratic precipitates in both eyes. The real-time polymerase chain reaction (RT-PCR) assay in the aqueous humor detected 98 copies/ml of chikungunya virus RNA. The patient underwent clear corneal phacoemulsification with in-the-bag intraocular lens implantation in the left eye with a good visual outcome. This is the first report where the presence of chikungunya virus RNA has been associated with a case of bilateral Fuchs’ heterochromic iridocyclitis.
Chikungunya virus; Fuchs’ heterochromic iridocyclitis; real-time polymerase chain reaction
We report a case of Usher syndrome in association with unilateral Fuchs’ heterochromic uveitis.
Fuchs’ heterochromic uveitis; Usher syndrome; deafness; blindness
Fuchs heterochromic uveitis (FHU) in its classic presentation is a unilateral, chronic, low grade, often asymptomatic anterior uveitis. It is characterized by a classic triad of iris heterochromia, cataract and keratic precipitates. Neovascularization of the iris and the anterior chamber (AC) angle (radial and circumferential) occurs in 6–22% of cases. This angle and iris new vessels can sometimes lead to a characteristic filiform haemorrhage and formation of hyphaema after AC paracentesis and is a hallmark of FHU known as Amsler–Verrey sign. This haemorrhage has been previously associated with trivial trauma, mydriasis, applanation tonometry, gonioscopy, or it may occur spontaneously. In the setting of cataract surgery it has been previously reported with the use of a Honan balloon. We report a case of cataract and FHU where the Amsler–Verrey sign developed intraoperatively during a phacoemulsification procedure. To the best of our knowledge, this is the first documented report of this sign occurring intraoperatively during cataract surgery.
The purpose of this study is to look for any possible associations in 58 consecutive cases of Fuchs heterochromic iridocyclitis (FHI) in a South Indian patient population. Fifty-eight consecutive cases (59 eyes) of FHI underwent a detailed ocular and systemic evaluation. Routine laboratory investigations for uveitis including serum angiotensin-converting enzyme and enzyme-linked immunosorbent assay (ELISA) for toxoplasmosis (IgG and IgM) were done in all the cases. Syndrome Evaluation System comprising of multiplex nucleic acid amplification and signature specific hybridization on the aqueous fluid was done in all 59 eyes for herpes simplex virus (HSV), varicella zoster virus, cytomegalovirus (CMV), rubella virus, chikungunya virus, Toxoplasma, and Mycobacterium tuberculosis. The results were statistically assessed using the SPSS (version 15) package.
Thirty-three males and 25 females with FHI were included in the study. Systemic sarcoidosis was seen in two cases. Serological tests failed to confirm an association with toxoplasmosis in all the cases. Aqueous fluid analysis showed positivity only to HSV (one case), CMV (one case), and chikungunya virus (one case).
We do see associations of sarcoidosis, HSV, and CMV in FHI in our patient populations as well. The detection of chikungunya virus in a patient with FHI in our series adds to the list of associations with FHI.
Implantation of iris-claw Artisan intraocular lens (IOL) is a surgical option for correction of aphakia; however, these IOLs have not been used in eyes with uveitis including Fuchs’ heterochromic iridocyclitis (FHI) due to possible risk of severe postoperative intraocular inflammation. In the case reported here, we secondarily implanted an Artisan IOL in a 28-year-old man with FHI who had aphakia with no capsular support due to a previous complicated cataract surgery. Enclavation was easily performed and no intraoperative complication was noted. Postoperative course was uneventful with no significant anterior chamber inflammation during 12 months of follow-up. Although there were few deposits on the IOL surface, the patient achieved a best-corrected visual acuity of 20/20 without developing glaucoma or other complications. Therefore, Artisan IOL may be considered for correction of aphakia in patients with FHI. However, studies on large number of patients are required to evaluate safety of the procedure.
Aphakia; Fuchs’ heterochromic iridocyclitis; iris-claw Artisan intraocular lens
Eighteen eyes in 17 patients with Fuchs' heterochromic iridocyclitis underwent cataract extraction with or without intraocular lens implantation (17 extracapsular and one intracapsular). Intraoperative complications included hyphaema, poor pupillary dilatations, and localised zonule dehiscence with vitreous loss. Only four eyes developed a marked anterior uveitus (two pseudophakic and two aphakic) which resolved within 2 weeks with topical steroids. Three eyes developed a rise in intraocular pressure (IOP) to more than 30 mm Hg on the first postoperative day. In all three eyes the IOP returned to normal off all therapy within 1 week. In one of three eyes preoperative glaucoma was made worse following surgery. Visual acuity testing revealed that 15 eyes (83.3%) achieved 6/12 vision or better. Lamellar macular hole, pre-existing macular scar, and pre-existing retinal detachment accounted for the poor visual result.
One pair, and probably two pairs, of monozygotic twins are reported with discordance for Fuchs' heterochromic uveitis (FHU). Regular Mendelian inheritance of this disease is now proved to be impossible. The heritability of FHU is low and may be zero. The possibility of any genetic predisposition to the disease and its association with 'simple' heterochromia are discussed.