Runx1 Orchestrates Sphingolipid Metabolism and Glucocorticoid Resistance in Lymphomagenesis.

The three-membered RUNX gene family includes RUNX1, a major mutational target in human leukemias, and displays hallmarks of both tumor suppressors and oncogenes. In mouse models, the Runx genes appear to act as conditional oncogenes, as ectopic expression is growth suppressive in normal cells but drives lymphoma development potently when combined with over-expressed Myc or loss of p53. Clues to underlying mechanisms emerged previously from murine fibroblasts where ectopic expression of any of the Runx genes promotes survival through direct and indirect regulation of key enzymes in sphingolipid metabolism associated with a shift in the "sphingolipid rheostat" from ceramide to sphingosine-1-phosphate (S1P). Testing of this relationship in lymphoma cells was therefore a high priority. We find that ectopic expression of Runx1 in lymphoma cells consistently perturbs the sphingolipid rheostat, whereas an essential physiological role for Runx1 is revealed by reduced S1P levels in normal spleen after partial Cre-mediated excision. Furthermore, we show that ectopic Runx1 expression confers increased resistance of lymphoma cells to glucocorticoid-mediated apoptosis, and elucidate the mechanism of cross-talk between glucocorticoid and sphingolipid metabolism through Sgpp1. Dexamethasone potently induces expression of Sgpp1 in T-lymphoma cells and drives cell death which is reduced by partial knockdown of Sgpp1 with shRNA or direct transcriptional repression of Sgpp1 by ectopic Runx1. Together these data show that Runx1 plays a role in regulating the sphingolipid rheostat in normal development and that perturbation of this cell fate regulator contributes to Runx-driven lymphomagenesis. J. Cell. Biochem. 118: 1432-1441, 2017. © 2016 Wiley Periodicals, Inc.

The effects of pre-slaughter restraint (for the purpose of cattle identification) on post-slaughter responses and carcass quality following the electrical stun/killing of cattle in a Jarvis Beef stunner.

This study compared normal post-Jarvis stun/kill responses and carcass quality with those occurring when crush restraint was not used during pre-slaughter. The carcasses of 1065 cattle slaughtered during one week at a commercial abattoir were evaluated for quality. The post-stun/kill responses of 788 of these animals were also assessed. An additional study of data from the carcasses of 6061 cattle was further evaluated for quality findings. A significant reduction in post-stun/kill limb movement, muscle tone and the expression of brainstem functions was recorded when restraint was not used. Abolishing crush restraint pre-slaughter also produced a significant reduction in the incidence of blood splash. In addition, the study also showed that animal identification post-slaughter could be successfully implemented with no negative consequences to food safety or traceability. It is suggested that abolishing the use of pre-slaughter crush restraint of cattle would enhance animal welfare and operator safety in plants whether electrical, or mechanical stunning was employed.

Head-only electrical stunning of poultry using a waterbath: a feasibility study.

1. The objective of this work was to investigate the feasibility of head only waterbath stunning as a means of generating immediate and long lasting unconsciousness while preventing wing flapping and avoiding carcass damage. 2. EEG measurements showed that immersion of the heads of the broilers for one second in a waterbath containing water of conductivity 2 x 5 mS/cm and a 50 Hz electric field of 10 V/cm resulted in immediate unconsciousness, and that increasing the electric field strength extended the duration of unconsciousness. 3. The passage of a 25-30 mA alternating current of frequency 2000 Hz through the broilers' bodies suppressed the wing flapping that followed a stun. 4. When the body current and electric field were applied simultaneously, wing flapping was prevented and EEG signals were suppressed for over 30 seconds indicating that the immediate unconsciousness lasted long enough to facilitate humane slaughter.

Broiler carcass quality using head-only electrical stunning in a waterbath.

1. The objective was to assess carcass quality of broilers when they were stunned by immersing their heads in a waterbath with an electric current flowing from one side of it to the other, while a second small current passed through the body to the waterbath to prevent involuntary wing flapping. 2. The prevalence of wing, shoulder and breast fillet haemorrhages and of broken bones in the pectoral region was not greater than that resulting from the normal stunning practice in that plant (63 mA, 610 Hz pDC). 3. These results imply that carcass damage using this technique will be significantly lower than that which will result from the application of higher stunning currents required by the new EU slaughter poultry slaughter regulations.

RUNX1 and its fusion oncoprotein derivative, RUNX1-ETO, induce senescence-like growth arrest independently of replicative stress.

A role for the RUNX genes in cancer fail-safe processes has been suggested by their induction of senescence-like growth arrest in primary murine fibroblasts and the failure of RAS-induced senescence in Runx2-deficient cells. We now show that RUNX1 induces senescence in human primary fibroblasts. High-affinity DNA binding is necessary but not sufficient, as shown by the functional attenuation of the truncated RUNX1/AML1a isoform and the TEL-RUNX1 fusion oncoprotein. However, a similar phenotype was potently induced by the RUNX1-ETO (AML1-ETO) oncoprotein, despite its dominant-negative potential. A detailed comparison of H-RAS(V12), RUNX1 and RUNX1-ETO senescent phenotypes showed that the RUNX effectors induce earlier growth stasis with only low levels of DNA damage signaling and a lack of chromatin condensation, a marker of irreversible growth arrest. In human fibroblasts, all effectors induced p53 in the absence of detectable p14(Arf), whereas only RUNX1-ETO induced senescence in p16(Ink4a)-null cells. Correlation was noted between induction of p53, reactive oxygen species and phospho-p38, whereas p38(MAPK) inhibition rescued cell growth markedly. These findings indicate a role for replication-independent pathways in RUNX and RUNX1-ETO senescence, and show that the context-specific oncogenic activity of RUNX1 fusion proteins is mirrored in their distinctive interactions with fail-safe responses.

The Jarvis Beef Stunner: Effects of a prototype chest electrode.

The Jarvis Beef Stunner electrically induces a stun, cardiac arrest and spinal discharge in adult cattle by three consecutive cycles. Concerns over varying incidences of cardiac arrest and some meat quality problems prompted this study to investigate the effect of replacing the conventional brisket electrode (T1) with a prototype chest electrode, with (T2), and without (T3) spinal discharge in 287 animals in a commercial abattoir. Repositioning the electrode did not significantly affect electrical parameters in any of the cycles. All animals received a cardiac arrest as assessed by electrocardiogram. Most post-stun/kill responses were unaffected. No difference was found in the incidence of a broken femur between treatments, but haemorrhages in the sirloin were significantly reduced by the prototype electrode (left: 51.1% (T1) vs. 30.3% (T3); right: 57.6% (T1) vs. 36.4% (T3)). The chest electrode was therefore efficient at causing cardiac arrest, did not significantly affect post-stun/kill responses and reduced haemorrhaging in the high-value sirloin.

Gene array analysis reveals a common Runx transcriptional programme controlling cell adhesion and survival.

The Runx genes are important in development and cancer, where they can act either as oncogenes or tumour suppressors. We compared the effects of ectopic Runx expression in established fibroblasts, where all three genes produce an indistinguishable phenotype entailing epithelioid morphology and increased cell survival under stress conditions. Gene array analysis revealed a strongly overlapping transcriptional signature, with no examples of opposing regulation of the same target gene. A common set of 50 highly regulated genes was identified after further filtering on regulation by inducible RUNX1-ER. This set revealed a strong bias toward genes with annotated roles in cancer and development, and a preponderance of targets encoding extracellular or surface proteins, reflecting the marked effects of Runx on cell adhesion. Furthermore, in silico prediction of resistance to glucocorticoid growth inhibition was confirmed in fibroblasts and lymphoid cells expressing ectopic Runx. The effects of fibroblast expression of common RUNX1 fusion oncoproteins (RUNX1-ETO, TEL-RUNX1 and CBFB-MYH11) were also tested. Although two direct Runx activation target genes were repressed (Ncam1 and Rgc32), the fusion proteins appeared to disrupt the regulation of downregulated targets (Cebpd, Id2 and Rgs2) rather than impose constitutive repression. These results elucidate the oncogenic potential of the Runx family and reveal novel targets for therapeutic inhibition.

Effects of salinity and the use of shackle/leg sprays on the electrical conductivity of a commercial waterbath stunner for broilers.

The effect of salinity and temperature on the electrical conductivity of a waterbath stunner was assessed in a commercial broiler processing plant. During a break in production, the waterbath was emptied and filled with either cold water, cold water plus sodium chloride, warm water, or warm water plus sodium chloride. Different concentrations of sodium chloride in distilled water at different temperatures were prepared in the laboratory and their conductivities were compared with the results obtained in the processing plant. No significant increase in conductivity was achieved through an increase in water temperature in either the laboratory or the processing plant, but there was an increase in conductivity after the addition of sodium chloride. However, the increase in conductivity was lost after 20 to 30 minutes of birds passing through the waterbath. The effects of the use, the location and the water temperature of the shackle/leg sprays on electrical conductivity were also studied, but under the conditions of the experiment none of these factors increased the electrical conductivity.

Killing of animals for disease control purposes.

In emergency disease outbreaks, large numbers of animals may need to be killed for control, containment and/or eradication purposes. Strategies for disease control will usually involve complete depopulation of herds/flocks, and may extend to depopulation across designated areas. Live animals present the major risk of spreading infectious agents so their slaughter should be completed as quickly as possible, with minimal pain and distress to the animals. The method chosen for killing of animals must be humane, efficient and safe for people and the environment. The tasks should be conducted under the supervision of an official veterinarian, supported by a sufficient number of adequately trained and competent personnel. It is essential to ensure all animals are dead, as demonstrated by the cessation of cardiac and respiratory movements. This paper discusses methods by which animals may be killed for disease control purposes.

Influence of specialization on the management and outcome of patients with pancreatic cancer.

BACKGROUND: Cancer care is increasingly specialized. Relationships between pancreatic cancer care, mortality and patterns of clinical practice among the full spectrum of patients, including those with irresectable tumours, are not well understood. METHODS: A cohort of 782 patients diagnosed prospectively with pancreatic cancer in 29 acute hospitals in England and Wales over 1 year were followed for 2-3 years. The effects of doctors' and hospitals' specialization, indicated by annual patient volumes, on operative mortality rates, survival times, and test and treatment provision were studied. Multiple logistic and Cox regression models were used to control for prognostic factors and treatments, providing adjusted odds and hazard ratios associated with a higher volume of ten patients annually. RESULTS: Patients managed by higher-volume hospitals survived significantly longer (hazard ratio 0.88 (95 per cent confidence interval (c.i.) 0.83 to 0.93); P < 0.001). They were more likely to undergo cytological examination (odds ratio (OR) 1.21 (95 per cent c.i. 1.01 to 1.35)), resection (OR 1.44 (1.17 to 1.79)) and biliary stenting (OR 1.17 (1.02 to 1.34)), and were less likely to have bypass surgery (OR 0.66 (0.55 to 0.78)). Patients of higher-volume doctors were more likely to undergo endoscopic retrograde cholangiopancreatography (OR 1.59 (1.19 to 2.11)), percutaneous transhepatic cholangiography (OR 1.50 (1.12 to 2.00)), laparoscopy (OR 1.81 (1.07 to 3.06)), resection (OR 1.84 (1.29 to 2.61)) and bypass surgery (1.71 (1.25 to 2.33)). CONCLUSION: Specialization appears to improve survival and to promote more thorough investigation.

Effect of frequency of the stunning current waveform on carcase and meat quality of turkeys processed in a commercial plant in the UK.

1. This study compared the effects of electrical stunning delivered using high and low frequency waveforms on carcase and meat quality of turkeys processed under commercial conditions. 2. The use of a high frequency waveform (1400 Hz) resulted in a faster bleedout and an improvement in carcase quality associated with a substantial reduction in haemorrhagic downgrading conditions. 3. Frequency of the applied waveform influenced breast muscle pH and colour although the magnitude of the differences was considered to be of minimal significance in influencing consumer perception.

Cohort study in South and West England of the influence of specialization on the management and outcome of patients with oesophageal and gastric cancers.

BACKGROUND: To evaluate specialization in National Health Service (NHS) cancer care, volume-outcome relationships were examined. METHODS: This was a cohort study of 1512 patients with oesophageal or gastric cancer in 23 acute NHS hospitals. Outcomes were survival time and operative (30 day) mortality. Multiple regression analysis was performed, adjusted for diagnoses, prognoses and treatments. RESULTS: For oesophageal cancer, the operative mortality rate decreased by 40 per cent (odds ratio 0.60 (95 per cent confidence interval (c.i.) 0.36 to 0.99 per cent); P = 0.047) for each increase of ten patients in doctors' annual surgical caseloads, and the risk of death decreased by 8 per cent (hazard ratio 0.92 (95 per cent c.i. 0.85 to 0.99); P = 0.021) for each increase of ten patients in doctors' annual caseloads. For gastric cancer, the operative mortality rate decreased by 41 per cent (odds ratio 0.59 (95 per cent c.i. 0.32 to 1.07)) for each increase of ten patients in doctors' annual surgical caseloads, and the risk of death decreased by 7 per cent (hazard ratio 0.93 (95 per cent c.i. 0.89 to 0.98); P = 0.009) for each increase of ten patients in hospitals' annual caseloads. Patients of higher-volume doctors were more likely to receive most investigations and treatments, independently of presenting features. CONCLUSION: The study supports concentration of services for oesophageal and gastric cancers. Specialization of doctors and their teams is at least as important as specialization of hospitals.

Electrical stunning of pigs: the effect of applied voltage on impedance to current flow and the operation of a fail-safe device.

The concept of a 'fail-safe' device to ensure that sufficient current is delivered to render a pig immediately unconscious when electrically stunned was investigated. For live pigs, no significant correlation could be determined between the pre-stun low voltage sensed impedance and the actual, higher voltage stun impedance. In contrast, a good correlation was found using heads from pigs killed more than 24 h previously. The impedance of a live pig's head was predominantly a function of the stunning voltage and decreased non-linearly with increasing voltage. The 'difference' between live and dead pigs was attributed to an 'ageing effect' which is not significant in the first few hours post mortem. No change in phase angle between corresponding stunning current and voltage waveforms, indicated that tissue reactance was not a contributing factor. It is concluded that 'fail-safe' devices based on low voltage pre-stun sensing are unlikely to meet the current legislative requirements (Council Directive 93/119/EC).

Commercial slaughter methods used on Atlantic salmon: determination of the onset of brain failure by electroencephalography.

This study investigated the effect of exsanguination without prior stunning, or carbon dioxide narcosis followed by exsanguination, or percussive stunning or spiking the brain, on the time taken to abolish the visual evoked responses (VERS) of farmed Atlantic salmon. Only percussive stunning and spiking killed the fish immediately and the other two methods resulted in aversive reactions by the fish. The VERS were lost between 148 and 440 seconds after exsanguination without stunning, and between 300 and 554 seconds after carbon dioxide narcosis followed by exsanguination. During both these procedures the fish showed strong aversive behaviour. In contrast, percussive stunning and spiking the brain could result in the immediate loss of VERS and no aversive reactions from the fish if the stun was applied correctly.

Electrical stunning of cattle.

Cattle are normally stunned electrically by three sequential cycles, first a three-second head-only cycle, to stun the animal, secondly a 15-second cardiac cycle, to induce ventricular fibrillation (cardiac arrest), and thirdly a four-second discharge cycle, to reduce convulsions after death. An effective and immediate stun was produced when > or =1.15 amps sinusoidal AC at 50 Hz was applied between the nose and neck electrodes for less than one second. However, when applied for three seconds, head-only currents of >0.46 amp sinusoidal AC at 50 Hz were sufficient to induce epileptiform activity in the brain, identified as high amplitude low frequency activity in the electroencephalogram. The induction of effective head-only electrical stunning resulted in an average interval of 50 seconds before the return of rhythmic breathing movements, and positive corneal and palpebral reflexes. The cardiac arrest cycle successfully induced ventricular fibrillation when >1.51 amps sinusoidal AC at 50 Hz was applied for five seconds between the nose and brisket electrodes.

Effectiveness of different electrical stunning regimens for turkeys and consequences for carcase quality.

This study examined the effectiveness of electrical stunning of turkeys applied at 150 mA per bird utilising a range of waveform-frequency combinations. In addition, the effect of stunning treatment on subsequent carcase quality was examined. The effectiveness of stunning, as judged by time to recovery, was similar regardless of bird weight or applied stunning treatment. Increasing waveform frequency was associated with a decrease in the prevalence of ventricular fibrillation, although turkeys appeared more susceptible than broilers. Use of the higher frequency waveforms (500 and 1500 Hz) was associated with a marked improvement in carcase quality, particularly with regard to breast muscle haemorrhaging and their use may result in considerable commercial advantage.

Effect of very low pulsed direct currents at high frequency on the return of neck tension in broilers.

Broiler chickens were stunned electrically for one second or 10 seconds with a unipolar pulsed direct current (DC) at 550 Hz. The effectiveness of the stun was assessed from the birds' behaviour. All the birds were stunned effectively when more than 15 mA true root mean square DC was applied for either period. An increase in the duration of the applied current resulted in an increased duration of unconsciousness. However, the unipolar pulsed DC at 550 Hz did not induce cardiac arrest, and it must therefore be accompanied by a prompt and effective neck cut, severing both carotid arteries.

Type IX collagen immunoreactive peptides in synovial fluids from arthritis patients.

OBJECTIVES: To determine whether type IX collagen-related peptides can be detected in the synovial fluids of arthritis patients and to assess their potential as molecular markers of arthritis. PATIENTS/METHODS: Synovial fluids from a set of carefully diagnosed arthritis patients and from healthy volunteers were used. Hydroxyproline assays were carried out to determine the content and concentration of collagen. Collagen cross-link determinations were conducted by reversed-phase HPLC. SDS PAGE and immunoblotting were used to identify the collagenous components, and N-terminal sequencing was performed to confirm these identities. RESULTS: All the synovial fluids were found to contain measurable amounts of collagen at similar concentrations. This appeared to be mainly high-molecular-weight material consisting of type I and type IX collagens, but not type II collagen. However, other smaller molecular weight type IX immunoreactive peptides were detected which were more apparent in the synovial fluids from arthritis patients. These peptides were also found to contain non-collagenous material. Collagen cross-links were also present in the arthritis synovial fluids. CONCLUSION: Collagenous material can be detected in all synovial fluids and the presence of pyridinoline cross-links indicates that at least some of this is derived from a mature collagen matrix. Type IX immunoreactive peptides were identified, but were found to contain significant amounts of non-collagenous material, and their presence, even at lower levels, in synovial fluids from normal subjects limits their potential for use as molecular markers of disease. Nevertheless, this is the first report of type IX collagen-related fragments in synovial fluids.

Effectiveness of electrical stunning applied using a variety of waveform-frequency combinations and consequences for carcase quality in broiler chickens.

1. The effectiveness of electrical stunning of poultry was assessed where the recommended current of 105 mA per bird was applied using a range of waveform-frequency combinations. In addition the effect of the treatments on subsequent carcase quality was investigated. 2. The use of frequencies greater than 50 Hz was associated with a failure to induce ventricular fibrillation. 3. All waveform-frequency combinations were equally effective in producing a stunned state; the time to recovery was not affected by stunning treatment but was significantly longer in heavier birds. 4. The use of high frequency (500 Hz and 1500 Hz) stunning currents resulted in a decrease in carcase downgrading and a marked reduction in the occurrence of breast muscle haemorrhages, which represent significant commercial benefits to be gained by their use.