Lupinosis in sheep. 1. Laboratory studies

IN earlier articles by Bennetts (1957, 1960), the clinical and pathological features of lupinosis, as seen in field outbreaks, were described. The suggestion was made that the roughage portion of the lupin plants was responsible for the disease. More recent observations suggest a relationship between parasitism and lupinosis

Use of insulin to increase epiblast cell number: towards a new approach for improving ESC isolation from human embryos

Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage.Jared M. Campbell, Michelle Lane, Ivan Vassiliev, and Mark B. Nottl

Toxic ferns of Western Australia

Botanical descriptions and notes on the distribution of six species of ferns are given. These plants contain the enzyme thiaminase, which may induce thiamin deficiency, or other constituents toxic to animals. The clinical signs of both thiamin deficiency and fern toxicity in affected livestock are described

Development of an improved porcine embryo culture medium for cloning, transgenesis and embryonic stem cell isolation

Work in our laboratory for more than two decades has focussed on the production of genetically modified pigs for xeno transplantion research. More recent work has focussed on the isolation of porcine embryonic stem cells to facilitate this as well as and other research applications. Central to this research has been the production of in vitro Produced (IVP) embryos. These embryos are produced using a twostep culture system based on NCSU23. This culture system which was developed by modifying energy substrate availability and concentrations and by adding non-essential and essential amino acids in a sequential manner. As a result of this work we have developed a culture system that better suits the changing metabolic needs of the pig embryo and produces embryos with relatively high developmental competence compared to the original formulation. These embryos can be used for a range of research applications including the isolation of embryonic stem cells.Luke FS Beebe, Stephen M McIlfatrick, Ivan M Vassiliev and Mark B Nottl

Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.Mark B. Nottle, Evelyn J. Salvaris, Nella Fisicaro, Stephen McIlfatrick, Ivan Vassiliev, Wayne J. Hawthorne, Philip J. O’Connell, Jamie L. Brady, Andrew M. Lew and Peter J. Cowa

Multipotent cell types in primary fibroblast cell lines used to clone pigs using somatic cell nuclear transfer

We have previously demonstrated that the use of porcine mesenchymal stem cells (MSCs) isolated from the bone marrow can increase the proportion of somatic cell nuclear transfer (SCNT) embryos that develop to the blastocyst stage compared with adult fibroblasts obtained from the same animal. The aim of the present study was to determine if MSCs are also present in primary cultures of adult fibroblasts which are commonly used for cloning live animals. To do this we chose a primary culture of adult fibroblasts that we had previously used to clone pigs. Single cell clones were isolated using low-density plating. After seven days of culture 63% of colonies displayed typical fibroblast morphology, while the remainder appeared cobblestone-like in appearance. Two of the 57 clones that displayed fibroblast morphology differentiated into adipocytes but not chondrocytes or osteocytes (uni-potent clones). Three of the 33 cobblestone-like clones differentiated into chondrocytes only, while 3 differentiated into adipocytes and chondrocytes but not osteocytes (bi-potent clones). One of the bi-potent cobblestone-like clones was then used for SCNT and in vitro development compared with a fibroblast-like clone which did not differentiate. Both cell types produced blastocysts at similar rates. In conclusion we have identified uni-potent and bi-potent cell types in primary cultures of adult fibroblasts used previously to clone live piglets.Sharon J. Harrison, Luke F.S. Beebe, Ivan Vassiliev, Stephen M. McIlfatrick and Mark B. Nottl

Control of IBMIR in Neonatal Porcine Islet Xenotransplantation in Baboons

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification

Insulin increases epiblast cell number of in vitro cultured mouse embryos via the PI3K/GSK3/p53 pathway

High-quality embryos give rise to embryonic stem cells (ESCs) at greater efficiencies than poor-quality embryos. However, most embryos available for human ESC derivation are of a reduced quality as a result of culture in relatively simple media up to 10 years earlier, before cryopreservation, or before compaction. In the present study, we used a mouse model to determine whether a culture with insulin from the 8-cell stage could increase the number of ESC progenitor epiblast cells in blastocysts, as well as endeavor to determine the molecular mechanism of the insulin's effect. Culture in media containing 1.7 ρM insulin increased epiblast cell number (determined by Oct4 and Nanog co-expression), and proportion in day 6 blastocysts. The inhibition of phosphoinositide 3 kinase (PI3K) (via LY294002), an early second messenger of the insulin receptor, blocked this effect. The inhibition of glycogen synthase kinase 3 (GSK3) or p53, 2 s messengers inactivated by insulin signaling (via CT99021 or pifithrin-α, respectively), increased epiblast cell numbers. When active, GSK3 and p53 block the transcription of Nanog, which is important for maintaining pluripotency. A simultaneous inhibition of GSK3 and p53 had no synergistic effects on epiblast cell number. The induced activation of GSK3 and p53, via the inhibition of proteins responsible for their inactivation (PKA via H-89 and SIRT-1 via nicotinamide, respectively), blocked the insulin's effect on the epiblast.From our findings, we conclude that insulin increases epiblast cell number via the activation of PI3K, which ultimately inactivates GSK3 and p53. Furthermore, we suggest that the inclusion of insulin in culture media could be used as a strategy for increasing the efficiency with which the ESC lines can be derived from cultured embryos.Jared M. Campbell, Mark B. Nottle, Ivan Vassiliev, Megan Mitchell, and Michelle Lan

Towards an integrated evaluation framework for xai: an experimental study

Increasing prevalence of opaque black-box AI has highlighted the need for explanations of their behaviours, for example, via explanation artefacts/proxy models. The current paper presents a paradigm for human-grounded experiments to evaluate the relationship between explanation fidelity, human learning performance, understanding and trust in a black-box AI by manipulating the complexity of an explanatory artefact. Decision trees were used in the current experiment as exemplar interpretable surrogate models, providing explanations approximating black-box behaviour, by means of explanation by simplification. Consistent with our hypotheses: 1) explanatory artefacts brought about better learning, while greater decision tree depths led to greater interpretability of the AI's performance and greater trust in the AI; and 2) explanatory artefacts facilitated learning and task performance even after they were withdrawn. Findings are discussed in terms of the interplay between human understanding, trust and AI system performance, highlighting the simplifying assumption of a monotonic relationship between explanation fidelity and interpretability

Modulation in voluntary neural drive in relation to muscle soreness

The aim of this study was to investigate whether (1) spinal modulation would change after non-exhausting eccentric exercise of the plantar flexor muscles that produced muscle soreness and (2) central modulation of the motor command would be linked to the development of muscle soreness. Ten healthy subjects volunteered to perform a single bout of backward downhill walking exercise (duration 30 min, velocity 1 ms−1, negative grade −25%, load 12% of body weight). Neuromuscular test sessions [H-reflex, M-wave, maximal voluntary torque (MVT)] were performed before, immediately after, as well as 1–3 days after the exercise bout. Immediately after exercise there was a −15% decrease in MVT of the plantar flexors partly attributable to an alteration in contractile properties (−23% in electrically evoked mechanical twitch). However, MVT failed to recover before the third day whereas the contractile properties had significantly recovered within the first day. This delayed recovery of MVT was likely related to a decrement in voluntary muscle drive. The decrease in voluntary activation occurred in the absence of any variation in spinal modulation estimated from the H-reflex. Our findings suggest the development of a supraspinal modulation perhaps linked to the presence of muscle soreness