Oncolytic adenovirus ORCA-010 increases the type 1 T cell stimulatory capacity of melanoma-conditioned dendritic cells.

Immune checkpoint blockade has resulted in durable responses in patients with metastatic melanoma, but only in a fraction of treated patients. For immune checkpoint inhibitors (ICI) to be effective, sufficient infiltration with tumor-reactive T cells is essential. Oncolytic viruses (OV) selectively replicate in and lyse tumor cells and so induce an immunogenic form of cell death, providing at once a source of tumor-associated (neo)antigens and of danger signals that together induce effective T cell immunity and tumor infiltration. Melanoma-associated suppression of dendritic cell (DC) differentiation effectively hampers OV- or immune checkpoint inhibitor (ICI)-induced anti-tumor immunity, due to a consequent inability to prime and attract anti-tumor effector T cells. Here, we set out to study the effect of ORCA-010, a clinical stage oncolytic adenovirus, on DC differentiation and functionality in the context of human melanoma. In melanoma and monocyte co-cultures, employing a panel of five melanoma cell lines with varying origins and oncogenic mutation status, we observed clear suppression of DC development with apparent skewing of monocyte differentiation to a more M2-macrophage-like state. We established the ability of ORCA-010 to productively infect and lyse the melanoma cells. Moreover, although ORCA-010 was unable to restore DC differentiation, it induced activation and an increased co-stimulatory capacity of monocyte-derived antigen-presenting cells. Their subsequent ability to prime effector T cells with a type I cytokine profile was significantly increased in an allogeneic mixed leukocyte reaction. Our findings suggest that ORCA-010 is a valuable immunotherapeutic agent for melanoma.

Characterization of acetylcholine release and the compensatory contribution of non-Ca(v)2.1 channels at motor nerve terminals of leaner Ca(v)2.1-mutant mice.

The severely ataxic and epileptic mouse leaner (Ln) carries a natural splice site mutation in Cacna1a, leading to a C-terminal truncation of the encoded Ca(v)2.1 alpha(1) protein. Ca(v)2.1 is a neuronal Ca(2+) channel, mediating neurotransmitter release at many central synapses and the peripheral neuromuscular junction (NMJ). With electrophysiological analyses we demonstrate severely reduced ( approximately 50%) neurotransmitter release at Ln NMJs. This equals the reduction at NMJs of Cacna1a null-mutant (Ca(v)2.1-KO) mice, which display a neurological phenotype remarkably similar to that of Ln mice. However, using selective Ca(v) channel blocking compounds we revealed a compensatory contribution profile of non-Ca(v)2.1 type channels at Ln NMJs that differs completely from that at Ca(v)2.1-KO NMJs. Our data indicate that the residual function and presence of Ln-mutated Ca(v)2.1 channels precludes presynaptic compensatory recruitment of Ca(v)1 and Ca(v)2.2 channels, and hampers that of Ca(v)2.3 channels. This is the first report directly showing at single synapses the deficits and plasticity in transmitter release resulting from the Ln mutation of Cacna1a.

The combined effects of grain supplementation and tenderstretching on alpaca (Vicugna pacos) meat quality.

This study investigated the effects of feeding a mixed grain supplement and tenderstretching (TS) alpaca carcasses on meat quality. A total of 56 castrated 24month old alpacas were divided into two treatments (pasture-only, and pasture plus supplementation). Supplemented groups were fed a mixed grain ration in addition to ad lib pasture for 10weeks. Animals were slaughtered across two kill days (n=28). One half of each carcass was suspended by the pelvis (TS) prior to chilling, and the other half was Achilles tendon hung (AH). After 24h, muscles were removed and aged for 10 and 25d. TS significantly increased sarcomere length and reduced shear force and cooking loss in the m. semimembranosus. This trend was not observed in other muscles including the m. longissimus thoracis et lumborum (LL) and m. psoas major. Ageing period resulted in a marginal improvement in LL tenderness. There is clear evidence that TS improves tenderness in the hindquarter of alpacas.

The impact of gender and age on the nutritional parameters of alpaca (Vicugna pacos) meat, colour stability and fat traits.

Meat quality parameters for 50 huacaya alpacas, representing three age groups (18, 24 and 36months) and two genders (females and castrated males) were studied. Prior to chilling, samples of m. longissimus thoracis et lumborum (LL) were taken to determine isocitrate dehydrogenase activity and glycogen concentration, and further meat quality samples were taken after 24h. Alpaca meat has low levels (<1%) of intramuscular fat in the LL, m. semimembranosus and m. biceps femoris, but the level in the LL increases with animal age, with levels of desirable polyunsaturated fatty acids (PUFA) ranging from 96.3 to 136.4mg/100g. Lipid oxidisation post retail display ranged from 1.27 to 1.96MDA/kg meat. Colour stability testing indicated alpaca meat was relatively stable during retail display with day of display having no significant effect on lightness (L*), or the 630nm/580nm ratio. These results may relate to the high proportion (76%) of glycolytic and type IIX muscle fibres in LL.

Post-weaning growth of beef heifers drenched with long- or short-acting anthelmintics.

OBJECTIVE: To determine whether (i) the drenching practices of beef producers in the Central Tablelands of NSW utilising short-acting anthelmintics limit the post-weaning growth of heifers and (ii) a single weaning treatment of a long-acting anthelmintic improves post-weaning growth. METHODS: Commencing at weaning, 20/100 heifers in each of five herds were injected four times at 90-day intervals with long-acting moxidectin to suppress gastrointestinal nematodes. In year 1 the balance was drenched using the owner's usual commercial practice with short-acting anthelmintics (5 herds) or left undrenched (1 herd). In year 2 the balance was drenched with long-acting moxidectin within 3 months of weaning (4 herds) or with short-acting drenches only (2 herds). At each visit live weight (LW) was recorded and a subsample tested for worm egg count (WEC) and plasma pepsinogen. RESULTS: In year 1 by 3 and 6 months' post weaning, suppressed heifers were significantly heavier than the undrenched and short-acting groups, and by 12 months were 40 and 28 kg heavier, respectively. In year 2, the LW of the suppressed and long-acting heifers was not different, but by 3 and 6 months both groups were significantly heavier than the short-acting group and by 12 months were 22 kg heavier. CONCLUSION: The post-weaning growth of beef heifers treated with short-acting drenches as adopted by Tablelands beef producers was slower than heifers drenched suppressively or with a single long-acting drench at weaning.

Optimum number of technical replicates for the measurement of compression of lamb meat.

Up to six (average 4.63) replicate compression values were collected on cooked m. semimembranosus of lambs that had been raised at six sites across southern Australia (n=1817). Measurements on each sample were made with one of two Lloyd Texture analyser machines, with each machine having a 0.63 cm diameter plunger. Based on a log normal model with common variance on the log scale for within sample replicate results, estimates of the within sample variability of compression values were obtained, resulting in a quality control procedure for compression testing based on the coefficient of variation.

Effect of electrical stimulation and ageing period on alpaca (Vicugna pacos) meat and eating quality.

This study investigated the effects of using medium voltage (~300 V) electrical stimulation (ES) and ageing on alpaca meat. A total of 50 huacaya alpacas were distributed across three age groups (18, 24 and 36 months) and two genders (females and castrated males). At 24h post mortem the m. longissimus thoracis et lumborum (LL) and m. semimembranosus (SM) muscles were removed and aged for either 5 or 10 days. In comparison to non-ES samples, ES significantly reduced: LL purge values by 3.0% and LL shear force (SF) at 5 and 10 days (by 21.6N and ageing further reduced tenderness by 6.6N), and SM SF by 5.8 N with significant age effects observed in both ES and non-ES SM samples, such that SF increased by 0.53 N with each month increase in animal age. Consumers rated ES samples higher on tenderness, juiciness, flavour and overall rating. ES and ageing of alpaca carcasses are recommended.

Restraining FOXO3-dependent transcriptional BMF activation underpins tumour growth and metastasis of E-cadherin-negative breast cancer.

Loss of cellular adhesion leads to the progression of breast cancer through acquisition of anchorage independence, also known as resistance to anoikis. Although inactivation of E-cadherin is essential for acquisition of anoikis resistance, it has remained unclear how metastatic breast cancer cells counterbalance the induction of apoptosis without E-cadherin-dependent cellular adhesion. We report here that E-cadherin inactivation in breast cancer cells induces PI3K/AKT-dependent FOXO3 inhibition and identify FOXO3 as a novel and direct transcriptional activator of the pro-apoptotic protein BMF. As a result, E-cadherin-negative breast fail to upregulate BMF upon transfer to anchorage independence, leading to anoikis resistance. Conversely, expression of BMF in E-cadherin-negative metastatic breast cancer cells is sufficient to inhibit tumour growth and dissemination in mice. In conclusion, we have identified repression of BMF as a major cue that underpins anoikis resistance and tumour dissemination in E-cadherin-deficient metastatic breast cancer.

Gene dosage-dependent transmitter release changes at neuromuscular synapses of CACNA1A R192Q knockin mice are non-progressive and do not lead to morphological changes or muscle weakness.

Ca(v)2.1 channels mediate neurotransmitter release at the neuromuscular junction (NMJ) and at many central synapses. Mutations in the encoding gene, CACNA1A, are thus likely to affect neurotransmitter release. Previously, we generated mice carrying the R192Q mutation, associated with human familial hemiplegic migraine type-1, and showed first evidence of enhanced presynaptic Ca(2+) influx [Neuron 41 (2004) 701]. Here, we characterize transmitter release in detail at mouse R192Q NMJs, including possible gene-dosage dependency, progression of changes with age, and associated morphological damage and muscle weakness. We found, at low Ca(2+), decreased paired-pulse facilitation of evoked acetylcholine release, elevated release probability, and increased size of the readily releasable transmitter vesicle pool. Spontaneous release was increased over a broad range of Ca(2+) concentrations (0.2-5mM). Upon high-rate nerve stimulation we observed some extra rundown of transmitter release. However, no clinical evidence of transmission block or muscle weakness was found, assessed with electromyography, grip-strength testing and muscle contraction experiments. We studied both adult ( approximately 3-6 months-old) and aged ( approximately 21-26 months-old) R192Q knockin mice to assess effects of chronic elevation of presynaptic Ca(2+) influx, but found no additional or progressive alterations. No changes in NMJ size or relevant ultrastructural parameters were found, at either age. Our characterizations strengthen the hypothesis of increased Ca(2+) flux through R192Q-mutated presynaptic Ca(v)2.1 channels and show that the resulting altered neurotransmitter release is not associated with morphological changes at the NMJ or muscle weakness, not even in the longer term.

A comparison of technical replicate (cuts) effect on lamb Warner-Bratzler shear force measurement precision.

The Warner-Bratzler shear force (WBSF) of 335 lamb m. longissimus lumborum (LL) caudal and cranial ends was measured to examine and simulate the effect of replicate number (r: 1-8) on the precision of mean WBSF estimates and to compare LL caudal and cranial end WBSF means. All LL were sourced from two experimental flocks as part of the Information Nucleus slaughter programme (CRC for Sheep Industry Innovation) and analysed using a Lloyd Texture analyser with a Warner-Bratzler blade attachment. WBSF data were natural logarithm (ln) transformed before statistical analysis. Mean ln(WBSF) precision improved as r increased; however the practical implications support an r equal to 6, as precision improves only marginally with additional replicates. Increasing LL sample replication results in better ln(WBSF) precision compared with increasing r, provided that sample replicates are removed from the same LL end. Cranial end mean WBSF was 11.2 ± 1.3% higher than the caudal end.

A national audit of retail lamb loin quality in Australia.

A retail audit of lamb loin tenderness was conducted over a 12-month period to determine the variation in tenderness of Australian lamb. Tenderness was objectively measured using Warner-Bratzler (WB) shear force. Muscle pH and cooking loss were determined on all samples and colour was measured on a sub-sample of loins. A total of 909 midloins from retail butcher shops and supermarkets located in four Australian capital cities (Sydney, Canberra, Melbourne, and Perth) were evaluated at four sampling times (December 1997 and March, June, and October 1998). Overall, 20.3% of all midloins purchased had a WB shear force value above the threshold level of 5 kg. Generic samples from Melbourne butcher shops were similar for WB shear force on average to the generic samples from Canberra and Sydney, whereas those from Melbourne supermarkets had significantly (P<0.001) higher WB shear force and were in line with generic samples from Perth. In both Canberra and Perth, alliance (branded) lamb had a greater WB shear force (P<0.05) than generic lamb. No relationship was found between price per kg and shear force (r=0.02) for loins purchased in Sydney (n=220). Price per kg differed between months (P<0.001) and suburbs (P<0.001), but not between retail butcher shops and supermarkets. Of the midloins tested, 10.3% had a pH above the critical point of 5.8. Midloins from the December 1997 sampling had a lower pH (P<0.01) than those sampled at other months. Those sampled in Melbourne and Perth had a similar mean pH, which were lower (P<0.001) than Canberra and Sydney samples. The findings from this quality audit suggest that there is room to improve the tenderness of Australian lamb sold in the domestic market. A lamb eating quality assurance system, based on set protocols, is one approach that is currently being investigated in Australia to ensure the supply of consistently high eating quality lamb to consumers.

Comparison of rankings for lean meat based on results from a CT scanner and a video image analysis system.

Coopworth cross lambs born over three years were examined in this study. Differences between two machines; a computer tomography (CT) scanner and a VIAScan® system for the estimation of carcase lean weight in lamb carcases was examined. The CT scanner provided a significantly higher estimate of carcase lean. The rank correlation (0.84) between the CT scanner and the VIAScan® system for the prediction of carcase lean was significant, but there was a different ranking for carcase lean depending on which machine was used. This has important ramifications for the use of VIAScan® data in the New Zealand Sheep Improvement Ltd genetic programme.

The impact of supplementing lambs with algae on growth, meat traits and oxidative status.

The current study examined the effect of supplementing lambs with algae. Forty, three month old lambs were allocated to receive a control ration based on oats and lupins (n=20) or the control ration with DHA-Gold™ algae (~2% of the ration, n=20). These lambs came from dams previously fed a ration based on either silage (high in omega-3) or oats and cottonseed meal (OCSM: high in omega-6) at joining (dam nutrition, DN). Lamb performance, carcase weight and GR fat content were not affected by treatment diet (control vs algae) or DN (silage vs OSCM). Health claimable omega-3 fatty acids (EPA+DHA) were significantly greater in the LL of lambs fed algae (125±6mg/100g meat) compared to those not fed algae (43±6mg/100g meat) and this effect was mediated by DN. Supplementing with algae high in DHA provides a means of improving an aspect of the health status of lamb meat.

Evaluation of the Hennessy Grading Probe for use in lamb carcases.

An evaluation of the Hennessy Grading Probe (HGP) was undertaken using 557 lamb carcases for the measurement of subcutaneous fat depth (Fat C) and muscle depth (EMD; m. longissimus thoracis et lumborum (LL)) between the 12th/13th ribs. All carcases were probed on the moving chain at up to 10 carcases per min and data analysis was constrained to 320 carcases which were probed at the target site (over the greatest depth of the LL at the midpoint between the 12th and 13th ribs). Analysis of the data revealed wide variability between HGP and equivalent carcase measures, but probe type (versions GP4 and GP7) had minimal impact on the usefulness of the HGP. The HGP as used in the study does not appear to be a viable option for the Australian sheep processing industry.

The interrelationship between sensory tenderness and shear force measured by the G2 Tenderometer and a Lloyd texture analyser fitted with a Warner-Bratzler head.

A comparison of peak shear force results for a Lloyd texture analyser fitted with a Warner Bratzler type of shearing head and a G2 Tenderometer was undertaken using sheep meat. The G2 is a new version of the Tenderometer that uses an electric linear motor to compress the sample, but still retains the blunt wedge-shaped "tooth". Analysis of sheep meat samples (n=121) revealed that the average G2 Tenderometer shear force results were approximately 1.2 times those for the Lloyd based on the following model; Lloyd=1.561 Tenderometer(0.84). Both instruments explained low amounts of the variation (less than 20%) in the sensory traits tenderness and overall liking. The high values for the sensory traits indicate that a wider range of samples, including samples with lower sensory scores, is required to develop robust threshold estimates so that either instrument could be use as an auditing instrument for the processing industry.

Interrelationship between measures of collagen, compression, shear force and tenderness.

This study examined the relationship between collagen content as determined by hydroxyproline assay with other measures of connective tissue, shear force and tenderness in lamb muscle. Samples were taken from both m. longissimus lumborum (LL, loin) and the m. semimembranosus (SM, topside) of 99 lambs. Sensory tenderness and compression of the LL were not correlated to any measure of collagen or connective tissue, with one exception where compression was correlated (r=-0.28; P<0.05) to the percentage of connective tissue determined by imaging. Intramuscular fat (IMF) was linearly correlated (P<0.05) to sensory tenderness and compression, such that a 1% increase in IMF increased the tenderness score by 2.3±0.83 units and reduced compression by 0.73±0.21 N. There was no correlation between SM shear force and collagen concentration. The data suggest that measurement of collagen concentration did not explain the variation in shear force and sensory tenderness observed in the meat from lambs.

SmartStretch™ technology. III. The impact of medium voltage stimulation and SmartStretch™ technology on sheep topside (m. semimembranosus) meat quality traits under commercial processing conditions.

This study evaluated the interaction between medium voltage electrical stimulation, SmartStretch™ stretching and ageing treatments on key meat quality traits of hot boned sheep m. semimembranosus. Medium voltage stimulation reduced initial pH (P<0.001), but did not impact on other meat quality traits. There was a significant interaction between stretch treatment and ageing (P<0.001) for shear force such that samples which were both stretched and aged were the most tender. Sarcomere length was significantly (P<0.001) increased by SmartStretch™ treatment. Control (no stretching) resulted in greater (P<0.05) cooking loss, but there was significantly less purge loss (P<0.05). The ratio 630/580 nm and a* colour values at 0 and 5 days decreased at a significantly (P<0.05) slower rate when SmartStretch™ was applied. Overall medium voltage stimulation did not inhibit the effectiveness of the SmartStretch™ treatment. The SmartStretch™ treatment provided significant improvement in tenderness and the potential to increase meat display time.

Examination of the effect of ageing and temperature at rigor on colour stability of lamb meat.

A study of factors (ageing period, rigor temperature and vitamin E level) impacting on the colour stability of lamb m. longissimus thoracis et lumborum (LL) during 3 days of simulated retail display was undertaken. The LL were taken from 84 lambs from 3 slaughters. Slices of LL were measured fresh (24h post-mortem) or after ageing for 5 days in vacuum packaging. The oxy/met ratio (630/580 nm), declined with display time, and increased with increasing temperature at pH6.0. Redness (a*) values also declined with display time and a reduction in redness values was observed as LL pH at 24h post-mortem and/or pH at 18°C increased. There was no effect of ageing period or vitamin E level on the oxy/met ratio or a* values when the vitamin E level averaged 3.76 mg/kg LL. These results suggest that maximising vitamin E levels in lambs and achieving a moderate rate of pH decline will optimise colour stability irrespective of ageing period.

SmartStretch™ technology. I. Improving the tenderness of sheep topsides (m. semimembranosus) using a meat stretching device.

This study evaluated the effect of stretching hot-boned sheep topsides using a pre-production prototype device (SmartStretch™). To test this effect, 40 sheep from 3 consignments were assessed. Left and right topsides were collected pre-rigour and randomly allocated to one of four treatments; 0 days ageing+SmartStretch™, 0 days ageing+no stretch, 5 days ageing+SmartStretch™ and 5 days ageing+no stretch. Meat from the 0 days aged+no stretch treatment was the least tender and the 5 days ageing+SmartStretch™ treatment resulted in the most tender meat. The m. semimembranosus from topsides stretched using the SmartStretch™ prototype device had a lower cooking loss percentage (P<0.001) and longer sarcomeres (P<0.001) than non-stretched m. semimembranosus. There was no effect of SmartStretch™ on myofibrillar degradation measured using particle size analysis (PSA), but there was an ageing effect (P<0.001). The tenderness of stretched m. semimembranosus showed significant improvement over non-stretched m. semimembranosus.

SmartStretch™ technology. II. Improving the tenderness of leg meat from sheep using a meat stretching device.

This study evaluated the effect of stretching hot-boned sheep hindlegs from 40 sheep carcases, classified as mutton, using a prototype device (SmartStretch™). Left and right legs were collected pre-rigor and randomly allocated to one of four treatments; 0days ageing+SmartStretch™, 0days ageing+no stretch, 5days ageing+SmartStretch™ and 5days ageing+no stretch. There was a significant interaction between stretch treatment and ageing (P<0.05) for shear force of the m. biceps femoris such that stretched and aged samples were the most tender. By contrast stretched m. semimembranosus (SM) had a significantly (P<0.05) lower shear force only at 0days of ageing. Stretching produced longer sarcomeres (P<0.001) for both the SM and m. semitendinosus muscles. Myofibrillar degradation indicated by particle size analysis or histology was not affected by stretching, but there was an ageing effect (P<0.001). SmartStretch™ provided significant improvements in tenderness of the individual muscles.