Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

Mainstreaming climate change adaptation : An incremental approach to disaster risk management in Australia

In this paper we argue that rationalist ‘predict then act’ approaches to disaster risk management (DRM) policy promote unrealistic public expectations of DRM provisions, the avoidance of decision making by political elites, an over-reliance on technical expertise and engineering solutions to reducing exposure to natural events, and a reactive approach to DRM overall. We propose an alternative incrementalist approach that focuses on managing uncertainties rather than reducing them and building resilience not simply through the reduction of hazard exposure, but also through the ongoing reduction of community vulnerability, the explicit consideration of normative priorities, and more effective community engagement in climate risk debates.

Rethinking disaster risk management and climate change adaptation

Australian governments face the twin challenges of dealing with extreme weather-related disasters (such as floods and bushfires) and adapting to the impacts of climate change. These challenges are connected, so any response would benefit from a more integrated approach across and between the different levels of government.This report summarises the findings of an NCCARF-funded project that addresses this problem. The project undertook a three-way comparative case study of the 2009 Victorian bushfires, the 2011 Perth Hills bushfires, and the 2011 Brisbane floods. It collected data from the official inquiry reports into each of these events, and conducted new interviews and workshops with key stakeholders. The findings of this project included recommendations that range from the conceptual to the practical. First, it was argued that a reconceptualization of terms such as ‘community’ and ‘resilience’ was necessary to allow for more tailored responses to varying circumstances. Second, it was suggested that the high level of uncertainty inherent in disaster risk management and climate change adaptation requires a more iterative approach to policymaking and planning. Third, some specific institutional reforms were proposed that included: 1) a new funding mechanism that would encourage collaboration between and across different levels of government, as well as promoting partnerships with business and the community; 2) improving community engagement through new resilience grants run by local councils; 3) embedding climate change researchers within disaster risk management agencies to promote institutional learning, and; 4) creating an inter-agency network that encourages collaboration between organisations.

The challenge of integrating climate change adaptation and disaster risk management : lessons from bushfire and flood inquiries in an Australian context

Emergency management and climate change adaptation will increasingly challenge all levels of government because of three main factors. First, Australia is extremely vulnerable to the impacts of climate change, particularly through the increasing frequency, duration and/or intensity of disasters such as floods and bushfires. Second, the system of government that divides powers by function and level can often act as a barrier to a well-integrated response. Third, policymaking processes struggle to cope with such complex inter-jurisdictional issues. This paper discusses these factors and explores the nature of the challenge for Australian governments. Investigations into the 2009 Victorian bushfires, the 2011 Perth Hills bushfires, and the 2011 Brisbane floods offer an indication of the challenges ahead and it is argued that there is a need to: improve community engagement and communication; refocus attention on resilience; improve interagency communication and collaboration; and, develop institutional arrangements that support continual improvement and policy learning. These findings offer an opportunity for improving responses as well as a starting point for integrating disaster risk management and climate change adaptation policies. The paper is based on the preliminary findings of an NCCARF funded research project: The Right Tool for the Job: Achieving climate change adaptation outcomes through improved disaster management policies, planning and risk management strategies involving Griffith University and RMIT. It should be noted from the outset that the purpose of this research project is not to criticise the actions of emergency service workers and volunteers who do an incredible job under extreme circumstances, often risking their own lives in the process. The aim is simply to offer emergency management agencies the opportunity to step back and rethink their overall approach to the challenge they face in the light of the impacts of climate change.

Public policy, disaster risk management and climate change adaptation

Public policymakers are caught in a dilemma : there is a growing list of urgent issues to address, at the same time that public expenditure is being cut. Adding to this dilemma is a system of government designed in the 19th century and competing theories of policymaking dating back to the 1950s. The interlinked problems of disaster risk management and climate change adaptation are cases in point. As the climate changes, there will be more frequent, intense and/or prolonged disasters such as floods and bushfires. Clearly a well integrated whole of government response is needed, but how might this be achieved? Further, how could academic research contribute to resolving this dilemma in a way that would produce something of theoretical interest as well as practical outcomes for policymakers? These are the questions addressed by our research via a comparative analysis of the 2009 Victorian bushfires, the 2011 Perth Hills bushfires, and the 2011 Brisbane floods. Our findings suggest that there is a need to: improve community engagement and communication; refocus attention on resilience; improve interagency communication and collaboration; and, develop institutional arrangements that support continual improvement and policy learning. These findings have implications for all areas of public policy theory and practice.

Rethinking emergency management and climate adaptation policies

Emergency management and climate change adaptation will increasingly challenge all levels of government because of three main factors. First, Australia is extremely vulnerable to the impacts of climate change, particularly through the increasing frequency and/or intensity of disasters such as floods and bushfires. Second, the system of government that divides powers by function and level can often act as a barrier to a well-integrated response. Third, policymaking processes struggle to cope with such complex inter-jurisdictional issues. This paper discusses these factors and explores the nature of the challenge for Australian governments. Investigations into the 2009 Victorian bushfires, the 2011 Perth Hills bushfires, and the 2011 Brisbane floods offer an indication of the challenges ahead and it is argued that there is a need to: improve community engagement and communication; refocus attention on resilience; improve interagency communication and collaboration; and, develop institutional arrangements that support continual improvement and policy learning. These findings offer an opportunity for improving responses as well as a starting point for integrating disaster risk management and climate change adaptation policies. The paper is based on the preliminary findings of an NCCARF funded research project: The Right Tool for the Job – Achieving climate change adaptation outcomes through improved disaster management policies, planning and risk management strategies involving Griffith University and RMIT.

Towards networked governance : improving interagency communication and collaboration for disaster risk management and climate change adaptation in Australia

Major disasters, such as bushfires or floods, place significant stress on scarce public resources. Climate change is likely to exacerbate this stress. An integrated approach to disaster risk management (DRM) and climate change adaptation (CCA) could reduce the stress by encouraging the more efficient use of pooled resources and expertise. A comparative analysis of three extreme climate-related events that occurred in Australia between 2009 and 2011 indicated that a strategy to improve interagency communication and collaboration would be a key factor in this type of policy/planning integration. These findings are in accord with the concepts of Joined-up Government and Network Governance. Five key reforms are proposed: developing a shared policy vision; adopting multi-level planning; integrating legislation; networking organisations; and establishing cooperative funding. These reforms are examined with reference to the related research literature in order to identify potential problems associated with their implementation. The findings are relevant for public policy generally but are particularly useful for CCA and DRM.

Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of V600EBRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of V600EBRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by V600EBRAF/MEK signalling in melanoma cells.

Gene regulation by translational inhibition is determined by Dicer partnering proteins

MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment1,​2,​3,​4. In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition1. In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript5. However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown6. Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized7,​8,​9, but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic systems.

Advanced tissue engineering scaffold design for regeneration of the complex hierarchical periodontal structure

AIM: This study investigated the ability of an osteoconductive biphasic scaffold to simultaneously regenerate alveolar bone, periodontal ligament and cementum. MATERIALS AND METHODS: A biphasic scaffold was built by attaching a fused deposition modelled bone compartment to a melt electrospun periodontal compartment. The bone compartment was coated with a calcium phosphate (CaP) layer for increasing osteoconductivity, seeded with osteoblasts and cultured in vitro for 6 weeks. The resulting constructs were then complemented with the placement of PDL cell sheets on the periodontal compartment, attached to a dentin block and subcutaneously implanted into athymic rats for 8 weeks. Scanning electron microscopy, X-ray diffraction, alkaline phosphatase and DNA content quantification, confocal laser microscopy, micro computerized tomography and histological analysis were employed to evaluate the scaffold's performance. RESULTS: The in vitro study showed that alkaline phosphatase activity was significantly increased in the CaP-coated samples and they also displayed enhanced mineralization. In the in vivo study, significantly more bone formation was observed in the coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted vascularization of the cell sheets, and periodontal attachment was achieved at the dentin interface. CONCLUSIONS: This work demonstrates that the combination of cell sheet technology together with an osteoconductive biphasic scaffold could be utilized to address the limitations of current periodontal regeneration techniques.

Biofabrication of customized bone grafts by combination of additive manufacturing and bioreactor knowhow

This study reports on an original concept of additive manufacturing for the fabrication of tissue engineered constructs (TEC), offering the possibility of concomitantly manufacturing a customized scaffold and a bioreactor chamber to any size and shape. As a proof of concept towards the development of anatomically relevant TECs, this concept was utilized for the design and fabrication of a highly porous sheep tibia scaffold around which a bioreactor chamber of similar shape was simultaneously built. The morphology of the bioreactor/scaffold device was investigated by micro-computed tomography and scanning electron microscopy confirming the porous architecture of the sheep tibiae as opposed to the non-porous nature of the bioreactor chamber. Additionally, this study demonstrates that both the shape, as well as the inner architecture of the device can significantly impact the perfusion of fluid within the scaffold architecture. Indeed, fluid flow modelling revealed that this was of significant importance for controlling the nutrition flow pattern within the scaffold and the bioreactor chamber, avoiding the formation of stagnant flow regions detrimental for in vitro tissue development. The bioreactor/scaffold device was dynamically seeded with human primary osteoblasts and cultured under bi-directional perfusion for two and six weeks. Primary human osteoblasts were observed homogenously distributed throughout the scaffold, and were viable for the six week culture period. This work demonstrates a novel application for additive manufacturing in the development of scaffolds and bioreactors. Given the intrinsic flexibility of the additive manufacturing technology platform developed, more complex culture systems can be fabricated which would contribute to the advances in customized and patient-specific tissue engineering strategies for a wide range of applications.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10-5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10-4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10-9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity

To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of 3 genome-wide association studies (GWAS) and 2 independent data sets genotyped on the Immunochip, including 10,588 cases and 22,806 controls. We identified 15 new susceptibility loci, increasing to 36 the number associated with psoriasis in European individuals. We also identified, using conditional analyses, five independent signals within previously known loci. The newly identified loci shared with other autoimmune diseases include candidate genes with roles in regulating T-cell function (such as RUNX3, TAGAP and STAT3). Notably, they included candidate genes whose products are involved in innate host defense, including interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C) and nuclear factor (NF)-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense. © 2012 Nature America, Inc. All rights reserved.