Modelling network travel time reliability under stochastic demand

A technique is proposed for estimating the probability distribution of total network travel time, in the light of normal day-to-day variations in the travel demand matrix over a road traffic network. A solution method is proposed, based on a single run of a standard traffic assignment model, which operates in two stages. In stage one, moments of the total travel time distribution are computed by an analytic method, based on the multivariate moments of the link flow vector. In stage two, a flexible family of density functions is fitted to these moments. It is discussed how the resulting distribution may in practice be used to characterise unreliability. Illustrative numerical tests are reported on a simple network, where the method is seen to provide a means for identifying sensitive or vulnerable links, and for examining the impact on network reliability of changes to link capacities. Computational considerations for large networks, and directions for further research, are discussed

Proteomic characterisation reveals active Wnt-signalling by human multipotent stromal cells as a key regulator of beta cell survival and proliferation

Aims/hypothesis: Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration. Methods: Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSCR) vs nonregenerative (MSCNR) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSCNR samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro. Results: MSCR showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSCNR uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSCR maintained Wnt signalling via Wnt5A/B (~2.5-fold increase) autocrine activity during ex vivo culture, while MSCNR repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSCNR samples increased the accumulation of nuclear β-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets. Conclusions/interpretation: Maintenance of active Wnt signalling within human MSCs promotes the secretion of matricellular and proangiogenic proteins that formulate a niche for islet regeneration

Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions

Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDHhi) stimulate islet regeneration after transplantation into mice with streptozotocin-induced β cell deletion. However, ALDHhi cells represent a rare progenitor subset and widespread use of UCB ALDHhi cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDHhi cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDHhi cells diminished as culture time progressed such that total ALDHhi cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDHlo subset) from less differentiated progeny with high ALDH-activity (ALDHhi subset). The ALDHhi subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34+/CD38- cells, 37.0% ± 6.9% CD34+/CD133+ cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDHlo subset. Notably, bulk cells or ALDHlo cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDHhi subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ

Expansion of Umbilical Cord Blood Aldehyde Dehydrogenase Expressing Cells Generates Myeloid Progenitor Cells that Stimulate Limb Revascularization

Uncompromised by chronic disease-related comorbidities, human umbilical cord blood (UCB) progenitor cells with high aldehyde dehydrogenase activity (ALDHhi cells) stimulate blood vessel regeneration after intra-muscular transplantation. However, implementation of cellular therapies using UCB ALDHhi cells for critical limb ischemia, the most severe form of severe peripheral artery disease, is limited by the rarity (\u3c0.5%) of these cells. Our goal was to generate a clinically-translatable, allogeneic cell population for vessel regenerative therapies, via ex vivo expansion of UCB ALDHhi cells without loss of pro-angiogenic potency. Purified UCB ALDHhi cells were expanded \u3e18-fold over 6-days under serum-free conditions. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, only 15.1% ± 1.3% of progeny maintained high ALDH-activity after culture. However, compared to fresh UCB cells, expansion increased the total number of ALDHhi cells (2.7-fold), CD34+/CD133+ cells (2.8-fold), and hematopoietic colony forming cells (7.7-fold). Remarkably, injection of expanded progeny accelerated recovery of perfusion and improved limb usage in immunodeficient mice with femoral artery ligation-induced limb ischemia. At 7 or 28 days post-transplantation, mice transplanted with expanded ALDHhi cells showed augmented endothelial cell proliferation and increased capillary density compared to controls. Expanded cells maintained pro-angiogenic mRNA expression and secreted angiogenesis-associated growth factors, chemokines, and matrix modifying proteins. Coculture with expanded cells augmented human microvascular endothelial cell survival and tubule formation under serum-starved, growth factor-reduced conditions. Expanded UCB-derived ALDHhi cells represent an alternative to autologous bone marrow as an accessible source of pro-angiogenic hematopoietic progenitor cells for the refinement of vascular regeneration-inductive therapies. Stem Cells Translational Medicine 2017;6:1607–1619

Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function

Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDH hi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDH hi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDH hi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDH hi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDH hi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased \u3e70-fold, the frequency of cells that retained ALDH hi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDH hi /CD34+ cell number by ≥ 10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB-inhibition of ALDH-activity delayed hematopoietic differentiation and expanded multipotent myeloid cells that accelerated vascular regeneration following i.m. transplantation in vivo. Stem Cells 2018;36:723–736

A novel archaeal regulatory protein, Sta1, activates transcription from viral promoters

While studying gene expression of the rudivirus SIRV1 in cells of its host, the hyperthermophilic crenarchaeon Sulfolobus, a novel archaeal transcriptional regulator was isolated. The 14 kDa protein, termed Sulfolobus transcription activator 1, Sta1, is encoded on the host chromosome. Its activating effect on transcription initiation from viral promoters was demonstrated in in vitro transcription experiments using a reconstituted host system containing the RNA polymerase, TATA-binding protein (TBP) and transcription factor B (TFB). Most pronounced activation was observed at low concentrations of either of the two transcription factors, TBP or TFB. Sta1 was able to bind viral promoters independently of any component of the host pre-initiation complex. Two binding sites were revealed by footprinting, one located in the core promoter region and the second ∼30 bp upstream of it. Comparative modeling, NMR and circular dichroism of Sta1 indicated that the protein contained a winged helix–turn–helix motif, most probably involved in DNA binding. This strategy of the archaeal virus to co-opt a host cell regulator to promote transcription of its genes resembles eukaryal virus–host relationships

Sheep Updates 2003 - Plenaries

This session covers eight papers from different authors:1. Biosecurity in the sheep industry will improve production and profits Ashley Mercy, Department of Agriculture, Western Australia 2. Easy care sheep David Scobie, AgResearch, PO Box 60 Lincoln, 8152, New Zealand 3. Targeting the market’s requirements - live sheep exports Neil Buchanan, Manager Animal Health Operations, Primary Industries & Resources, South Australia 4. Setting flock breeding objectives Kevin Bell, Sheep Management and Production Consultants, Kojonup WA 5. Future feeding of sheep in Western Australia John Milton and Graeme Martin, The University of Western Australia 6. Recipe for getting back into Sheep Bob Hall, JRL Hall and Co., Darkan 7. Wool producer experiences with individual animal management Maurie Stephen, Wool Producer, Armidale NSW 8. The history of future sheep worm management Dr Rob Woodgate, Department of Agriculture, Western Australi