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.

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.

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.

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.

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.

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.

Type IX collagen: a possible function in articular cartilage.

The effect of type IX on in vitro fibrillogenesis of type II collagen indicated that, while not preventing fibrillogenesis, the presence of type IX collagen reduced the size of the type II fibre aggregates. This observation is consistent with the in vivo localisation studies of type IX collagen. Using the immunogold labelling technique, type IX collagen was shown to be located evenly on small fibrils which occur at higher concentration closer to the cell. Therefore type IX collagen may function as a regulator of fibre diameter in articular cartilage.

Susceptibility of the cartilage collagens types II, IX and XI to degradation by the cysteine proteinases, cathepsins B and L.

We have investigated the susceptibility of both the helical and non-helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37 degrees C and pH values from 3.5 to 7.0. Cleavage occurs only within the non-helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

1991 Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: an immunolocalization study.

Several types of collagen are known to exist in the intervertebral disc in addition to the fibrillar collagens, Types I and II. Although they constitute only a small percentage of the total collagen content, these minor collagens may have important functions. This study was designed to investigate the presence of Types I, II, III, IV, VI, and IX collagens in the intervertebral disc and cartilage end plate by immunohistochemistry, thereby establishing their location within the tissues. Types III and VI collagen have a pericellular distribution in animal and human tissue. No staining for Type IX collagen was present in normal human disc, but in rat and bovine intervertebral disc, it was also located pericellularly. These results show that cells of the intervertebral disc and cartilage end plate sit in fibrous capsules, forming chondrons similar to those described in articular cartilage. In pathologic tissue the amount and distribution of the collagen types, and the organization of the pericellular capsule, differ from that seen in control material.

Effect of a 350 Hz DC stunning current on evoked responses in the chicken's brain.

Somatosensory evoked responses in the chicken's brain were examined before and after stunning with a 350 Hz pulsed DC. When the current was greater than 120 mA, 93 per cent of the birds lost their evoked responses for at least 60 seconds. On this basis 120 mA per bird is recommended as an acceptable current for commercial use.

Effect of depth of immersion in the waterbath on the effectiveness of electrical stunning in chickens.

Broiler chickens were electrically stunned either by immersing their heads, necks and upper breast in a waterbath stunner or by immersing their heads only. The time to recovery of muscular activity was assessed, and it was found that the deeper immersion was associated with a slightly (10 second) shorter time to resumption of head righting. When hens which had previously been implanted with electroencephalogram electrodes were electrically stunned it was found that the incidence of somatosensory evoked responses during the first 60 seconds after applying the current was not influenced by the depth of immersion. It was concluded that depth of immersion had little influence on the effectiveness of electrical stunning.

Turkey slaughtering procedures: time to loss of brain responsiveness after exsanguination or cardiac arrest.

The effect of inducing a cardiac arrest or severing both carotid arteries on the spontaneous and visual evoked electrical activity in the brain were examined in anaesthetised turkeys. The time to loss of evoked activity was greater following the cardiac arrest. It was concluded that the potential humanitarian advantages of inducing a cardiac arrest at stunning are less pronounced in the turkey than for other poultry species.

Pig slaughtering procedures: time to loss of brain responsiveness after exsanguination of cardiac arrest.

The time taken for a conventional chest stick to produce the loss of flash-evoked responsiveness in the electrocorticogram of anaesthetised pigs was compared with a method involving fibrillation of the heart. On average severing the brachiocephalic trunk and anterior vena cava took 18 seconds to induce a loss of brain responsiveness, and inducing cardiac fibrillation took 19 seconds. It is concluded that a pig slaughtering method that stuns the brain and at the same time fibrillates the heart is likely to be more humane than the conventional slaughtering methods where there is a delay between the stunning and sticking operations.

Effect of wetting a chicken's feathers on the effectiveness of electrical stunning.

Chickens were electrically stunned either when their bodies and feathers were wet or when they were dry. The time to recovery of muscular activity following stunning with 81 mA was assessed, and it was found that it was quicker in the wet birds. When hens which had previously been implanted with electroencephalogram electrodes were stunned with 119 mA it was found that the prevalence of somatosensory evoked responses during the first 60 seconds after applying the current was not significantly influenced by the wetness of the birds. It was concluded that electrical stunning with low currents in wet birds leads to a lower prevalence of ventricular fibrillation at stunning and a shorter lasting stun in the non-fibrillated birds. This effect is possibly due to some of the current tracking over the bird's wet body rather than through the brain.

Effect of incomplete immersion of the head in waterbath stunners on the effectiveness of electrical stunning in ducks.

Ten ducks were subjected to electrical stunning with their heads completely immersed in the water of a waterbath, and 10 ducks received the same current while only the bill and skin over the crop made contact with the water. The effect on visual evoked responses in the brain was examined. A higher proportion of birds had lost their visual evoked responses immediately following the current with whole head immersion. It is suggested that crop and bill immersion is less effective in disturbing brain function than whole head immersion.

Studies on the central nervous system: visually evoked cortical responses in sheep.

Several factors which were thought to affect the magnitude of flash evoked cortical potentials were examined in halothane-anaesthetised sheep. The proximity of the electrodes to the cortex and the patency of the eye which was contralateral to the recording electrodes had the greatest effect on the excursion distance of the visually evoked response. The choice of upper frequency filter (70 vs 700 Hz), the patency of the ipsilateral eye and closure of both eyelids resulted in relatively smaller reductions in excursion distance of the response. The importance of these findings in studies using evoked potentials for assessing sensibility and brain death in animals is discussed.

Time to loss of brain responsiveness following exsanguination in calves.

In the anaesthetised calf the time to loss of visually evoked cortical responsiveness after cutting the common carotid arteries and the jugular veins was 17 seconds. An earlier study showed that the corresponding interval for sheep was 14 seconds. In contrast to suggestions made by other workers, it is concluded that the time necessary for sticking at slaughter to induce brain dysfunction is similar in the two species.

Effect of carbon dioxide stunning on somatosensory evoked potentials in hens.

The spontaneous electroencephalogram (EEG) and somatosensory evoked potentials (SEPs) were examined in 17 hens before and during stunning in 45 per cent carbon dioxide. The results indicated that EEG suppression and loss of SEPs occurred in 21 +/- 4 s and 30 +/- 2 s, respectively, after exposure to carbon dioxide, eventually leading to EEG silence in 101 +/- 18 s. Convulsions occurred 15 seconds after the loss of SEPs, suggesting that the hens convulsed while they were unconscious. It is concluded that the induction of anaesthesia can be rapid in carbon dioxide stunning and in this respect carbon dioxide is potentially suitable for stunning poultry under commercial conditions.

Concussive methods of pre-slaughter stunning in sheep: assessment of brain function using cortical evoked responses.

Averaged cortical evoked responses were used to evaluate brain function in anaesthetised sheep. Effects on both evoked somatosensory responses (SERs) and visual evoked responses (VERs) were examined. Following capitive bolt stunning, SERs and VERs were abolished instantaneously and did not reappear for the duration of the experiment. Similar results were found when animals were shot while conscious. It was concluded that captive bolt stunning in sheep produces an immediate, profound and long lasting brain failure and is therefore an effective preslaughter stunning method.