Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Valproic acid selectively increases vascular endothelial tissue-type plasminogen activator production and reduces thrombus formation in the mouse.

Essentials Stimulating endogenous fibrinolysis could be a novel antithrombotic strategy. The effect of valproic acid on endothelial tissue plasminogen activator in mice was investigated. Valproic acid increased tissue plasminogen activator expression in vascular endothelium. Valproic acid reduced fibrin deposition and thrombus formation after vascular injury. SUMMARY: Background The endogenous fibrinolytic system has rarely been considered as a target to prevent thrombotic disease. Tissue-type plasminogen activator (t-PA) production is potently increased by histone deacetylase (HDAC) inhibitors in endothelial cells in vitro, but whether this translates into increased vascular t-PA production and an enhanced fibrinolytic capacity in vivo is unknown. Objectives To determine whether the HDAC inhibitor valproic acid (VPA) stimulates production of t-PA in the vasculature of mice, and whether VPA pretreatment affects fibrin deposition and clot formation after mechanical vessel injury. Methods Mice were injected with VPA twice daily for up to 5 days. t-PA mRNA, and antigen expression in the mouse aorta and the circulating levels of t-PA were determined. Fibrin and thrombus dynamics after mechanical vessel injury were monitored with intravital confocal microscopy. Potential effects of VPA on platelets and coagulation were investigated. Results and Conclusions We found that VPA treatment increased vascular t-PA production in vivo and, importantly, that VPA administration was associated with reduced fibrin accumulation and smaller thrombi in response to vascular injury, but still was not associated with an increased risk of bleeding. Furthermore, we observed that higher concentrations of VPA were required to stimulate t-PA production in the brain than in the vasculature. Thus, this study shows that VPA can be dosed to selectively manipulate the fibrinolytic system in the vascular compartment and reduce thrombus formation in vivo.

Survivability of Escherichia coli O157:H7 in mechanically tenderized beef steaks subjected to lactic acid application and cooking under simulated industry conditions.

Mechanical tenderization improves the palatability of beef; however, it increases the risk of translocating pathogenic bacteria to the interior of beef cuts. This study investigated the efficacies of lactic acid spray (LA; 5 % ), storage, and cooking on the survivability of Escherichia coli O157:H7 in mechanically tenderized beef steaks managed under simulated industry conditions. Beef subprimals inoculated with either high (10(5) CFU/ml) or low (10(3) CFU/ml) levels of E. coli O157:H7 were treated (LA or control) and stored for 21 days prior to mechanical tenderization, steak portioning (2.54 cm), and additional storage for 7 days. Steaks were then cooked to an internal temperature of 55, 60, 65, 70, or 75°C. Samples were enumerated and analyzed using DNA-based methods. Treatment with LA immediately reduced E. coli O157:H7 on the lean and fat surfaces of high- and low-inoculum-treated subprimals by more than 1.0 log CFU/cm(2) (P < 0.05). Storage for 21 days reduced surface populations of E. coli O157:H7 regardless of the inoculation level; however, the populations on LA- and control-treated lean surfaces of high- and low-inoculum-treated subprimals were not different after 21 days (P > 0.05). E. coli O157:H7 was detected in core samples from high-inoculum-treated steaks cooked to 55, 60, or 70°C. Conversely, E. coli O157:H7 was not detected in core samples from low-inoculum-treated steaks, regardless of the internal cooking temperature. These data suggest that LA- and storage-mediated reduction of pathogens on subprimals exposed to typical industry contamination levels (10(1) CFU/cm(2)) reduces the risk of pathogen translocation and subsequent survival after cooking.

Differentiating haemostasis from thrombosis for therapeutic benefit.

The central role of platelets in the formation of the primary haemostatic plug as well as in the development of arterial thrombosis is well defined. In general, the molecular events underpinning these processes are broadly similar. Whilst it has long been known that disturbances in blood flow, changes in platelet reactivity and enhanced coagulation reactions facilitate pathological thrombus formation, the precise details underlying these events remain incompletely understood. Intravital microscopy studies have highlighted the dynamic and heterogeneous nature of thrombus development and demonstrated that there are considerable spatiotemporal differences in the activation states of platelets within a forming thrombus. In this review we will consider the factors regulating the activation state of platelets in a developing thrombus and discuss how specific prothrombotic factors may influence this process, leading to excessive thrombus propagation. We will also discuss some potentially novel therapeutic approaches that may reduce excess thrombus development whilst minimising bleeding risk.

Storage length, storage temperature, and lean formulation influence the shelf-life and stability of traditionally packaged ground beef.

The effect of storage length and temperature on the shelf life of three ground beef formulations (lean:fat: 73:27, 81:19 and 91:9) was investigated. Coarsely ground beef was stored at -1.7 or 2.3°C for up to 28d. Traditional overwrap packages were produced every 7d prior to retail display for 24h. Lipid oxidation (TBARS), subjective color, instrumental color, and aerobic bacteria were evaluated after 0 and 24h of display. Formulation influenced initial L* and subjective color values (P<0.05). Storage temperature did not affect initial color, but product stored at 2.3°C was more discolored after 24h (P<0.05). Aerobic bacteria increased as storage d and temperature increased (P<0.05). Initial TBARS increased through d 21, but were lower after 28d. Overall, initial characteristics depended on formulation; however, ground beef shelf-life and stability were largely influenced by storage length and storage temperature.

The heritabilities, phenotypic correlations, and genetic correlations of lean color and palatability measures from longissimus muscle in beef cattle.

Data from a study conducted over 5 yr were analyzed to determine heritability estimates of LM lean color, as measured by subjective scoring and Hunter Colorimeter readings, and palatability, as measured by trained sensory panelists and Warner-Bratzler shear force (WBSF). Phenotypic and genetic correlations were determined between each of the measures of palatability and color. There were 1,066 cattle representing 12 different breeds in the study. Subjective lean color and a* (redness) and b* (yellowness) values were moderately heritable, 0.34 ± 0.122, 0.29 ± 0.115 and 0.28 ± 0.120, respectively, whereas the L* (lightness) was lowly heritable, 0.09 ± 0.087. The heritability of WBSF was moderately heritable ranging from 0.23 ± 0.114 (3 d) to 0.42 ± 0.148 (21 d). Sustained tenderness, as measured by sensory panelists, was found to be moderately heritable ranging from 0.16 ± 0.108 (21 d) to 0.33 ± 0.135 (14 d). Sustained juiciness and beef flavor, as measured by sensory panelists, were found to be lowly to moderately heritable ranging from 0.00 ± 0.089 (21 d) to 0.18 ± 0.105 (14 d) and 0.00 ± 0.080 (7 d) to 0.18 ± 0.110 (21 d), respectively. The significant phenotypic correlations were those between WBSF and subjective lean color, L* value, and a* value; both initial and sustained tenderness as well as beef flavor were correlated with subjective lean color and L* value. Flavor intensity and overall mouthfeel were associated with subjective lean color, L* value, a* value, and b* value. Both a* and b* values were highly correlated genetically with WBSF, -0.71 and -0.72, respectively, and subjective lean color was moderately correlated with WBSF, -0.46. The genetic correlation between subjective lean color and initial tenderness was also high, 0.56, whereas that between a* value and initial tenderness was 0.43, which was similar to that found between b* value and initial tenderness, 0.44. The genetic correlations between subjective lean color, a* value, and b* value with sustained tenderness were all high at 0.58, 0.70, and 0.58, respectively. The genetic correlations between a* value and b* value with beef flavor were low to moderate at 0.12 and 0.19, respectively, whereas that between subjective lean color and beef flavor was high, 0.64. The genetic correlations between a* value, b* value, and lean color with sustained juiciness were all moderate correlations at -0.35, -0.23, and -0.45, respectively. The genetic correlations between a* value and b* value with overall mouthfeel were high at 0.80 and 0.79, respectively, whereas that between subjective lean color and overall mouthfeel was moderate, 0.46. In conclusion, regardless of measurement technique of lean color, it was not only heritable but was also moderately to highly correlated with measurements of palatability in beef from LM.

Safety and efficacy of targeting platelet proteinase-activated receptors in combination with existing anti-platelet drugs as antithrombotics in mice.

BACKGROUND AND PURPOSE: Developing novel anti-platelet strategies is fundamental to reducing the impact of thrombotic diseases. Thrombin activates platelets via proteinase-activated receptors (PARs), and PAR antagonists are being evaluated in clinical trials for prevention of arterial thrombosis. However, one such trial was recently suspended due to increased bleeding in patients receiving a PAR1 antagonist in addition to anti-platelet drugs that most often included both aspirin and clopidogrel. Therefore, it remains unclear how to best manipulate PARs for safe antithrombotic activity. To address this, we have examined potential interactions between existing anti-platelet drugs and strategies that target PARs. EXPERIMENTAL APPROACH: We used in vivo mouse models in which interactions between various anti-platelet strategies could be evaluated. We examined the effects on thrombosis and haemostasis in PAR4 -/- mice (platelets unresponsive to thrombin) treated with therapeutic doses of either aspirin or clopidogrel. KEY RESULTS: Using a model in which occlusive thrombosis occurred in PAR4 -/- mice or wild-type mice treated with aspirin or clopidogrel, PAR4 -/- mice treated with either anti-platelet agent showed marked protection against thrombosis. This antithrombotic effect occurred without any effect on haemostasis with aspirin, but not clopidogrel. Furthermore, specifically targeting thrombin-induced platelet activation (via PARs) improved the therapeutic window of non-specifically inhibiting thrombin functions (via anticoagulants). CONCLUSIONS AND IMPLICATIONS: Our results indicate that PAR antagonists used in combination with aspirin provide a potent yet safe antithrombotic strategy in mice and provide insights into the safety and efficacy of using PAR antagonists for the prevention of acute coronary syndromes in humans.

Effects of high-oil corn on feedlot performance, carcass characteristics, fatty acid profiles, beef palatability, and retail case life traits of beef top loin steaks.

Our objective was to compare the effects of feeding steam-flaked, high-oil corn with normal steam-flaked corn to which yellow grease was added to equalize dietary fat on performance and carcass characteristics of finishing beef steers, and palatability, retail case life, and fatty acid composition of strip loins. Angus steers (n = 120; initial BW = 288 kg) were allotted to dietary treatments consisting of 1) normal mill-run, steam-flaked corn plus added fat (NMR) or 2) high-oil, steam-flaked corn (HOC) and assigned randomly to pens (12 pens/treatment with 5 steers/pen). Performance (ADG, DMI, and G:F) was measured over time, and cattle were shipped to a commercial abattoir for collection of carcass data after 165 d on feed. Carcass data were collected at 48 h postmortem on all carcasses, and 2 carcasses from each pen were selected randomly for collection of strip loins (IMPS #180A). At 14 d postmortem, 4 steaks (2.54 cm thick) were removed for retail display, trained sensory panel analysis, Warner-Bratzler shear force determination, and fatty acid analysis. Daily BW gain was greater (P = 0.03) and G:F was increased 8.4% (P = 0.01) for steers fed NMR compared with HOC, but DMI was not affected (P > 0.10) by treatment. No treatment differences were observed (P > 0.10) for HCW, 12th-rib fat, KPH, and yield grade. Marbling scores were greater (P = 0.01) for NMR than for HOC, and LM area tended (P = 0.07) to be greater in NMR than in HOC carcasses. The proportion of carcasses grading USDA Choice did not differ (P = 0.77) between treatments, but a greater (P = 0.04) proportion of carcasses graded in the upper two-thirds of Choice for NMR vs. HOC. Trained sensory panel traits and Warner-Bratzler shear force values did not differ between treatments (P > 0.10), and no differences (P > 0.10) were detected for purge loss or fatty acid composition. Overall, ADG and G:F were less and marbling score was decreased, but there were no differences between treatments in beef palatability, retail case life, or concentrations of fatty acids in strip loins.

Dynamics of platelet thrombus formation.

Platelet aggregation and thrombus formation at sites of atherosclerotic plaque rupture is a dynamic process that can lead to intermittent or permanent obstruction to blood flow, resulting in ischemic tissue injury and organ dysfunction. There is a growing body of evidence suggesting that the dynamics of platelet aggregation and initial thrombus development are regulated by two distinct, complementary processes, involving: (i) rheological (biomechanical) and (ii) soluble-agonist-dependent mechanisms. Rheological-dependent platelet aggregation occurs between discoid platelets and requires the biomechanical adhesive and signaling function (mechanotransduction) of the major platelet adhesion receptors, GPIb and integrin alpha(IIb)beta3. Soluble agonists further potentiate platelet activation, stimulating global platelet shape change and degranulation, and play a major role in stabilizing formed aggregates. Unraveling the dynamics of platelet aggregation and thrombus formation in vivo requires consideration of the cooperative interplay between rheological- and soluble agonist-dependent platelet aggregation mechanisms.

DNA damage responses at low radiation doses.

Increased cell killing after exposure to low acute doses of X rays (0-0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (<1 Gy) doses in hyper-radiosensitive cells and that at these doses the influence of RAD51-mediated repair events may increase, based on high levels of RAD51/BRCA2 repair foci. These data do not support a threshold model for activation of DNA repair in hyper-radiosensitive cells but do suggest that the balance of repair enzyme activity may change at low doses.

Patient attitudes to sternotomy and thoracotomy scars.

BACKGROUND: There are no data comparing patient attitudes to sternotomy and thoracotomy scars following surgery for congenital heart disease (CHD). METHODS: Two hundred and one patients with a scar from CHD surgery (105 sternotomy, 36 thoracotomy, and 60 both scars) had a structured interview to explore attitudes to their scar. RESULTS: Comparable proportions of each group reported that they did not like or hated their scar (23/105 [22 %] sternotomy, 9/36 [25 %] thoracotomy, 17/60 [28 %] both scars). Significantly more patients stated that they where embarrassed by and/or their choice of clothing was affected by a thoracotomy scar (20/36, 56 %) than those with a sternotomy scar (36/105, 34 %), p = 0.04. This was also seen when comparing sternotomy alone with both scars (36/105 [34 %] vs. 34/60 [57 %], p = 0.008). CONCLUSIONS: Adults who have undergone surgery for CHD are more likely to have a negative attitude to a thoracotomy than a sternotomy scar. Before a change in surgical approach is considered based on patient preferences, the acceptability and psychological impact of the different scars following surgery needs formal study.

Exploiting the DNA repair defect in BRCA mutant cells in the design of new therapeutic strategies for cancer.

Individuals harboring germ-line mutations in the BRCA1 or BRCA2 genes are at highly elevated risk of a variety of cancers. Ten years of research has revealed roles for BRCA1 and BRCA2 in a wide variety of cellular processes. However, it seems likely that the function of these proteins in DNA repair is critically important in maintaining genome stability. Despite this increasing knowledge of the defects present in BRCA-deficient cells, BRCA mutation carriers developing cancer are still treated similarly to sporadic cases. Here we describe our efforts, based on understanding the DNA repair defects in BRCAdeficient cells, to define the optimal existing treatment for cancers arising in BRCA mutation carriers and, additionally, the development of novel therapeutic approaches. Finally, we discuss how therapies developed to treat BRCA mutant tumors might be applied to some sporadic cancers sharing similar specific defects in DNA repair.

Human cell senescence as a DNA damage response.

It has been established that telomere-dependent replicative senescence of human fibroblasts is stress-dependent. First, it was shown that telomere shortening, which is a major contributor to telomere uncapping, is stress-dependent to a significant degree. Second, the signalling pathway connecting telomere uncapping and replicative senescence appears to be the same as the one that is activated by DNA damage: uncapped telomeres activate signalling cascades involving the protein kinases ATM, ATR and, possibly, DNA-PK. Furthermore, phosphorylation of histone H2A.X facilitates the formation of DNA damage foci around uncapped telomeres, and this in turn activates downstream kinases Chk1 and Chk2 and, eventually, p53. It appears that this signalling pathway has to be maintained in order to keep cells in a senescent state. Thus, cellular senescence can be regarded as a permanently maintained DNA damage response state. This suggests that antibodies against DNA damage foci components might be useful markers for senescent cells in vivo.

Life insurance and mortgage application in adults with congenital heart disease.

OBJECTIVE: To compare the outcome of life insurance and mortgage applications of adults with congenital heart disease (CHD) with controls and at different severities of CHD. METHODS: Two hundred and ninety-nine adult CHD patients underwent a questionnaire-based interview by a trained nurse. They were asked to give an identical questionnaire to a friend to act as a control. One hundred and seventy-seven controls replied. CHD patients were classified into three categories based on severity. Comparisons were made between matched controls and between different severities of CHD. RESULTS: Similar proportions of the CHD group (59%) had applied for life insurance as matched controls (56%). Compared to controls, significantly more of the adults with CHD who had applied for life insurance have been refused (34 vs 4%, P < 0.0001) or asked to pay extra (37 vs 6%, P = 0.0002). Mortgage application rate was also similar in both groups with more of the CHD patients refused than matched controls (20 vs 3%, P = 0.0004). These differences in both life insurance and mortgage remain significant when the cases and controls are matched by employment status and NYHA functional class. There was no significant difference in life insurance and mortgage application outcome between the groups of mild, significant and complex CHD. CONCLUSIONS: Adults with CHD are significantly more likely to have difficulty obtaining life insurance or a mortgage than controls. Refusal rates appear to be independent of the severity of CHD. This suggests that the label of CHD may have a negative impact despite the lesion being minor and that the outcome of an individual application is difficult to predict based on the severity of the CHD. The increasing numbers of adults with CHD suggest that this problem is likely to increase and needs to be addressed as it can have a major impact on the patient's quality of life.

Phosphoinositide 3-kinases and the regulation of platelet function.

A clear understanding of the role of PI (phosphoinositide) 3-kinases in supporting the haemostatic function of platelets has been slow to evolve. In fact, insight into the roles of individual PI 3-kinase isoforms in platelet function remains rudimentary. However, based on in vitro studies using wortmannin and LY294002, there is evidence for an important role for PI 3-kinases in regulating a broad range of functional platelet responses, including primary platelet adhesion, cytoskeletal remodelling and platelet aggregation. One of the critical platelet responses involves affinity regulation of the major platelet integrin alphaIIbbeta3, the primary receptor mediating platelet aggregation and thrombus growth. The input signals regulating integrin alphaIIbbeta3 can be divided into three main groups: (1) G(q)-coupled receptors linked to the activation of PLCbeta (phospholipase Cbeta); (2) G(i)-coupled receptors linked to the regulation of adenylate cyclase and Rap1b; and (3) adhesion receptor signalling involving Src kinase-dependent activation of PLCgamma isoforms. PI 3-kinases have not been demonstrated to play a critical role in G(q)-dependent platelet activation; however, one or more PI 3-kinase isoforms appears to be important for G(i)-dependent activation of Rap1b and adhesion receptor activation of PLCgamma isoforms. Thus distinct co-operative PI 3-kinase signalling mechanisms appear to play an important role in regulating the adhesive function of integrin alphaIIbbeta3.

Characterization and consumer acceptance of three muscles from Hampshire x Rambouillet cross sheep expressing the callipyge phenotype.

Eight Hampshire x Rambouillet crossbred wethers expressing the callipyge phenotype and eight Hampshire x Rambouillet half-sibling wethers with a normal phenotype were slaughtered when they reached 59 kg. The supraspinatus (SPM), longissimus (LM), and semitendinosus (STM) muscles were analyzed to determine callipyge effects on calpain and calpastatin activities, sarcomere length, percentage of muscle fiber types, and muscle fiber areas. After 14 d of aging, chops were frozen until analyses for trained sensory panel evaluations, Warner-Bratzler shear force values, and consumer perceptions of tenderness, flavor, juiciness, and overall satisfaction of chops were conducted. Calpastatin activity was 57% greater (P < 0.05) and m-calpain activity was 33% greater (P < 0.05) in muscles from carcasses of callipyge than normal sheep. Sarcomeres were shorter (P < 0.001) in the LM than the SPM or STM, regardless of phenotype. Muscle fiber area was 76% larger (P < 0.05) in the LM of callipyge than normal sheep, but muscle fiber area was not affected (P > 0.05) by phenotype in the SPM or STM. Phenotype had no effect (P = 0.12) on the percentage of slow-twitch, oxidative fiber types in any of the three muscles. In STM and LM from callipyge lambs, the percentage of fast-twitch, oxidative/glycolytic fibers was lower (P < 0.05) and that of fast-twitch-glycolytic fibers was higher (P < 0.05) than in their normal counterparts. Phenotype did not affect (P = 0.90) the fiber type percentage in the SPM. Callipyge LM were less tender and normal LM were more tender than other chops (P < 0.05). Callipyge loin chops had higher Warner-Bratzler shear force values than other chops (P < 0.001). Consumers rated fewer (P < 0.05) callipyge loin and shoulder chops acceptable in juiciness, tenderness, and overall acceptability than normal chops, but phenotype did not affect (P > 0.05) consumer acceptability of leg chops. These results indicate that LM from Hampshire x Rambouillet sheep displaying the callipyge phenotype had higher calpastatin activity and were less tender than the LM from normal sheep. In addition, consumer perceptions indicated that only one in 10 leg chops, one in five shoulder chops, and one in four loin chops from callipyge sheep were unacceptable.

Signaling events underlying thrombus formation.

Recent in vivo studies have highlighted the dynamic and complex nature of platelet thrombus growth and the requirement for multiple adhesive receptor-ligand interactions in this process. In particular, the importance of von Willebrand factor (VWF) in promoting both primary adhesion and aggregation under high shear conditions is now well established. In general, the efficiency with which platelets adhere and aggregate at sites of vessel wall injury is dependent on the synergistic action of various adhesive and soluble agonist receptors, with the contribution of each of the individual receptors dependent on the prevailing blood flow conditions. In this review, we will discuss the major platelet adhesive interactions regulating platelet thrombus formation under high shear, with specific focus on the VWF (GPIb and integrin alphaIIbbeta3) and collagen receptors (GPVI and integrin alpha2beta1). We will also discuss the signaling mechanisms utilized by these receptors to induce platelet activation with specific emphasis on the role of cytosolic calcium flux in regulating platelet adhesion dynamics. The role of soluble agonists in promoting thrombus growth will be highlighted and a model to explain the synergistic requirement for adhesive and soluble stimuli for efficient platelet aggregation will be discussed.

Localized morphoea preceded by a pigmented purpuric dermatosis.

Localized morphoea uncommonly occurs in a linear distribution and may present following trauma, although most cases are idiopathic. Pigmented purpuric dermatoses such as lichen aureus may also rarely occur in a linear distribution and have been associated with trauma. A middle-aged man is described who initially presented with lesions typical of lichen aureus in a linear distribution at a site exposed to chronic low-grade trauma. This eruption was transient and clinically underwent spontaneous complete resolution. Several months later he developed localized morphoea in an identical distribution. To our knowledge this is the first reported case of a pigmented purpuric dermatosis, presumably precipitated by trauma, evolving into linear morphoea.