An Enhanced Linkage Map of the Sheep Genome Comprising More Than 1000 Loci

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.     

Antifibrotic effects of antioxidant N-acetylcysteine in a mouse model of human hypertrophic cardiomyopathy mutation.

OBJECTIVES: The objective was to determine the effects of antioxidant N-acetylcysteine (NAC) on reversal and attenuation of established interstitial fibrosis in the cardiac troponin T (cTnT) mouse model of human hypertrophic cardiomyopathy (HCM) mutation. BACKGROUND: Interstitial fibrosis is a characteristic pathological feature of HCM and a risk factor for sudden cardiac death. The cTnT-Q92 transgenic mice, generated by cardiac-restricted expression of human HCM mutation, show a two- to four-fold increase in interstitial fibrosis. METHODS: We randomized the cTnT-Q92 mice to treatment with a placebo or NAC (250, 500, or 1,000 mg/kg/day) and included non-transgenic mice as controls (N = 5 to 13 per group). We performed echocardiography before and 24 weeks after therapy, followed by histologic and molecular characterization. RESULTS: There were no significant differences in the baseline characteristics of the groups. Treatment with NAC reduced myocardial concentrations of malondialdehyde and 4-hydroxy-2(E)-nonenal, markers of oxidative stress, by 40%. Collagen volume fractions comprised 1.94 +/- 0.76% of the myocardium in non-transgenic, 6.2 +/- 1.65% in the placebo, and 1.56 +/- 0.98% in the NAC (1,000 mg/kg/day) groups (p < 0.001). Expression levels of Col1a1 and Col1a2 were also reduced significantly, as were levels of phosphorylated but not total p44/42, p38, and c-Jun NH2-terminal kinase. Levels of oxidized mitochondrial and nuclear DNA were not significantly different. CONCLUSIONS: Treatment with NAC reduced myocardial oxidative stress, stress-responsive signaling kinases, and fibrosis in a mouse model of HCM. The potential beneficial effects of NAC in reversal of cardiac phenotype in human HCM, the most common cause of sudden cardiac death in the young, merits investigation.     

Predicting soil depth using simple ground-based measurements of stem shape and taper in the butt swell section of individual Pinus radiata trees

Predicting soil depth using simple ground-based measurements of the tree stem has multiple benefits for precision (site-specific) forest management and estimating carbon stocks of plantation forests. Current methods of mapping soil depth rely on collecting a sufficient density of direct soil measurements, which is expensive and typically not feasible over extensive forest areas. The availability of detailed soil depth information under forest plantations is consequently sparse and this presents a significant impediment to precision forest management and the ability to estimate forest soil carbon stocks. In this study, we propose that the relationship between stem shape and taper in the butt swell of individual Pinus radiata trees and soil depth can be described in a simple empirical model. We demonstrate that shape and taper of the butt-swell section of the tree stem are as robust predictors of soil depth as individual tree height, and also have the advantage of being easy to measure from the ground. This finding has potential benefits for reducing the cost of soil data collection and improving fine-scale forest soil mapping.     

Human Molecular Genetic and Functional Studies Identify TRIM63, Encoding Muscle RING Finger Protein 1, as a Novel Gene for Human Hypertrophic Cardiomyopathy

Rationale: A delicate balance between protein synthesis and degradation maintains cardiac size and function. TRIM63 encoding Muscle RING Finger 1 (MuRF1) maintains muscle protein homeostasis by tagging the sarcomere proteins with ubiquitin for subsequent degradation by the ubiquitin-proteasome system (UPS). Objective: To determine the pathogenic role of TRIM63 in human hypertrophic cardiomyopathy (HCM). Methods and Results: Sequencing of TRIM63 gene in 302 HCM probands (250 white individuals) and 339 control subjects (262 white individuals) led to identification of 2 missense (p.A48V and p.I130M) and a deletion (p.Q247*) variants exclusively in the HCM probands. These 3 variants were absent in 751 additional control subjects screened by TaqMan assays. Likewise, rare variants were enriched in the white HCM population (11/250, 4.4% versus 3/262, 1.1%, respectively, P=0.024). Expression of the mutant TRIM63 was associated with mislocalization of TRIM63 to sarcomere Z disks, impaired auto-ubiquitination, reduced ubiquitination and UPS-mediated degradation of myosin heavy chain 6, cardiac myosin binding protein C, calcineurin (PPP3CB), and p-MTOR in adult cardiac myocytes. Induced expression of the mutant TRIM63 in the mouse heart was associated with cardiac hypertrophy, activation of the MTOR-S6K and calcineurin pathways, and expression of the hypertrophic markers, which were normalized on turning off expression of the mutant protein. Conclusions: TRIM63 mutations, identified in patients with HCM, impart loss-of-function effects on E3 ligase activity and are probably causal mutations in HCM. The findings implicate impaired protein degradation in the pathogenesis of HCM.     

Pharmacokinetic and toxicity study of an intraocular cyclosporine DDS in the anterior segment of rabbit eyes

PURPOSE: To establish the safety and pharmacokinetic efficacy of an Oculex Drug Delivery System (DDS; Oculex Pharmaceuticals, Inc., Sunnyvale, CA) containing cyclosporin A (CsA) in the anterior segment of the rabbit eye. METHODS: The Oculex DDS is an intraocular, sustained-release, drug delivery system comprising a biodegradable lactic acid-glycolic acid copolymer. A controlled prospective study was performed that involved implanting a DDS containing 0.5 mg of CsA into the anterior chamber (AC) of the right eyes of 16 New Zealand White rabbits. A placebo DDS was implanted into the left eyes of these same rabbits as the control. Slit lamp examinations and AC taps were performed serially, and the rabbits were killed and the globes removed at 2, 4, 8, and 12 weeks for histology and determination of CsA drug levels. Analysis of CsA levels was performed with high-performance liquid chromatography-mass spectrometry. RESULTS: High concentrations of CsA were detectable in all layers of the cornea (epithelium, corneal stroma and endothelium) throughout the 3-month period. Low CsA levels were detected in the aqueous, whereas no CsA was detectable in the blood. There were no adverse reactions observed. CONCLUSIONS: The Oculex DDS CsA device is effective in delivering long-term levels of CsA to corneal tissues, without adverse effects. Further studies in an animal model of corneal transplant rejection should be performed to determine the potential of this device in the prophylaxis and treatment of corneal transplant rejection in humans.     

Effects of shock waves on microcirculation, perfusion, and pain management in critical limb ischemia

Shock waves (SW) are used to control pain in different clinical conditions (eg, painful knee, elbow, and shoulder, etc). The effects of SWs may be due to cellular ;;stunning' (particularly nervous components). It may also be the consequence of unknown metabolic actions on tissues, which may include changes in cellular permeability, the liberation of proteins and mediators locally acting on pain and nerve endings. The aim of this study was to evaluate the reduction in pain and the improvement in microcirculation induced by SW treatment in a 2-week study in patients with chronic limb ischemia (CLI). Of the 32 patients with CLI, 30 (20 with rest pain only, 10 with necrosis) completed the study. The treatment was well tolerated. Foot radiographs performed before and after treatment indicate no bone damage after treatment. Foot (tibial arteries) blood pressure was unchanged after 2 weeks. The increase in laser Doppler flux was significant (p <0.05) after treatment. The ORACLE score at 2 weeks was decreased (p <0.05). The same trend was observed with the analogue scale line for pain (p <0.05). Partial pressure of oxygen (PO2) increased (p <0.05) and partial pressure of carbon dioxide (PCO2) decreased (p <0.05). In all patients an increase in pain-free walking distance was observed (the distance increased on average 2.4 times). Flux improvement was still present after 1 month. The outcome at 3 months in these patients indicates that the improvement (concerning the survival of the limbs) was persistent. In conclusion SWs treatment in CLI produced changes both on the microcirculation and pain. These results are very interesting, confirming previous observations, and opening new treatment options in CLI. The skin flow improvement did not relate to an increase in pressure.     

Asiaticoside Inhibits TNF‐α‐Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells

The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor‐alpha (TNF‐α), a pro‐atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF‐α in human aortic endothelial cells (HAEC). TNF‐α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)‐dextran. Asiaticoside pretreatment significantly suppressed TNF‐α‐induced increased permeability. Asiaticoside also prevented TNF‐α‐induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF‐α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti‐hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D‐induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF‐α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F‐actin organization. Copyright © 2015 John Wiley & Sons, Ltd.     

Using a balloon applicator for intraoperative radiotherapy in laparoscopic resection of perforated upper rectal cancer

Techniques in Coloproctology -