Mitochondrial targeting of cyclosporin A enables selective inhibition of cyclophilin-D and enhanced cytoprotection after glucose and oxygen deprivation

CsA (cyclosporin A) is a hydrophobic undecapeptide that inhibits CyPs (cyclophilins), a family of PPIases (peptidylprolyl cis–trans isomerases). In some experimental models, CsA offers partial protection against lethal cell injury brought about by transient ischaemia; this is believed to reflect inhibition of CyP-D, a mitochondrial isoform that facilitates formation of the permeability transition pore in the mitochondrial inner membrane. To evaluate this further, we have targeted CsA to mitochondria so that it becomes selective for CyP-D in cells. This was achieved by conjugating the inhibitor to the lipophilic triphenylphosphonium cation, enabling its accumulation in mitochondria due to the inner membrane potential. In a cell-free system and in B50 neuroblastoma cells the novel reagent (but not CsA itself) preferentially inhibited CyP-D over extramitochondrial CyP-A. In hippocampal neurons, mitochondrial targeting markedly enhanced the capacity of CsA to prevent cell necrosis brought about by oxygen and glucose deprivation, but largely abolished its capacity to inhibit glutamate-induced cell death. It is concluded that CyP-D has a major pathogenic role in ‘energy failure’, but not in glutamate excitotoxicity, where cytoprotection primarily reflects CsA interaction with extramitochondrial CyPs and calcineurin. Moreover, the therapeutic potential of CsA against ischaemia/reperfusion injuries not involving glutamate may be improved by mitochondrial targeting

An investigation into alternative domestic water, sewer and electricity supply systems in the eThekwini municipal area.

Thesis (M.Sc.)-University of KwaZulu- Natal, Durban, 2004.Millions of Black South Africans still lack access to adequate housing, mainly as a result of apartheid era development policies. The delivery of low income, state subsidised, housing includes the provision of water, sewer drainage and electricity supply services. These services are provided via individual connections to the bulk infrastructure, or grid, supply network. Whilst this delivery mechanism meets community aspirations, it masks the environmental impact of this access to natural resources. This research investigates the low income housing delivery mechanism in South Africa, both past and present, and considers the associated infrastructural service delivery in the context of what is understood as sustainable development. In order to identify a more environmentally sustainable format of service delivery, the notion of autonomous housing is investigated. This investigative research establishes the body of knowledge in respect of rainwater harvesting and renewable energy sources capable of being harvested at a domestic level and uses this knowledge to inductively derive theoretical models for the provision of water and electricity supply as well as sewer drainage to low income housing in the Ethekwini Municipal area. The objective of the research is therefore to propose a more autonomous, or self reliant, system of service delivery that constitutes sustainable development

Applying a Modified Technology Acceptance Model to Qualitatively Analyse the Factors Affecting Microblogging Integration

The purpose of this research is to examine factors affecting students’ perception and engagement of microblogging integration using a qualitative approach. We employed a qualitative case study design to explore potential factors affecting microblogging integration in a hybrid course. Using the technology acceptance model (TAM) model as an umbrella framework, we examined through in-depth interviews with 18 participants the impact of microblogging integration into instruction that affected students’ reported use and perceptions of their microblogging-supported learning experiences. We found that individual differences, system characteristics, social influence and facilitating conditions all have impact on student participation and engagement in microblogging integration to varying degrees. We identified more granular factors within each of the four dimensions. Additionally, we proposed a Twitter user taxonomy based on perceived usefulness and usage behaviour and discussed its broad implications in higher education learning environments. Finally, we identified several pedagogical implications pertaining to strategies of microblogging integration under the context of a hybrid course and offered pertinent recommendations for future research

Methane emissions from cattle: estimates from short-term measurements using a Green Feed system compared with measurements obtained using respiration chambers or sulphur hexafluoride tracer

The Green Feed (GF) system (C-Lock Inc., Rapid City, USA) is used to estimate total daily methane emissions of individual cattle using short-term measurements obtained over several days. Our objective was to compare measurements of methane emission by growing cattle obtained using the GF system with measurements using respiration chambers (RC)or sulphur hexafluoride tracer (SF6). It was hypothesised that estimates of methane emission for individual animals and treatments would be similar for GF compared to RC or SF6 techniques. In experiment 1, maize or grass silage-based diets were fed to four growing Holstein heifers, whilst for experiment 2, four different heifers were fed four haylage treatments. Both experiments were a 4 × 4 Latin square design with 33 day periods. Green Feed measurements of methane emission were obtained over 7 days (days 22–28) and com-pared to subsequent RC measurements over 4 days (days 29–33). For experiment 3, 12growing heifers rotationally grazed three swards for 26 days, with simultaneous GF and SF6 measurements over two 4 day measurement periods (days 15–19 and days 22–26).Overall methane emissions (g/day and g/kg dry matter intake [DMI]) measured using GF in experiments 1 (198 and 26.6, respectively) and 2 (208 and 27.8, respectively) were similar to averages obtained using RC (218 and 28.3, respectively for experiment 1; and 209 and 27.7, respectively, for experiment 2); but there was poor concordance between the two methods (0.1043 for experiments 1 and 2 combined). Overall, methane emissions measured using SF6 were higher (P<0.001) than GF during grazing (186 vs. 164 g/day), but there was significant (P<0.01) concordance between the two methods (0.6017). There were fewer methane measurements by GF under grazing conditions in experiment 3 (1.60/day) com-pared to indoor measurements in experiments 1 (2.11/day) and 2 (2.34/day). Significant treatment effects on methane emission measured using RC and SF6 were not evident for GF measurements, and the ranking for treatments and individual animals differed using the GF system. We conclude that under our conditions of use the GF system was unable to detectsignificant treatment and individual animal differences in methane emissions that were identified using both RC and SF6techniques, in part due to limited numbers and timing ofmeasurements obtained. Our data suggest that successful use of the GF system is reliant on the number and timing of measurements obtained relative to diurnal patterns of methane emission

Cybersecurity Awareness Shrewsbury Public Schools

In the 21st Century, technology reaches every aspect of our lives. As “digital citizens” we must be aware of the dangers both to our technological equipment and our personal information stored, transmitted, and processed on this equipment. The Cybersecurity Awareness curriculum developed for the Shrewsbury Public School district is designed to meet this need, as well as foster an interest in technology and ethical computer use

A Proposal for Stewardship Support to Private Native Forests in NSW

The Southern Cross Group is proposing a completely new approach to private forest management in NSW based on stewardship support. This means that incentives will be used to encourage landholders to manage their forests in a way that maintains their environmental and other values for the community, without compromising their value as a resource to the farming community. Importantly, the Southern Cross Group system will foster good outcomes through innovation rather than through cumbersome and onerous prescriptions. Private forests in NSW are important as a source of timber as well as for the conservation values they provide to the general community. Landholders should be encouraged to manage them in a way that preserves their productive capacity and their conservation values for the long term. At present, however, neither the current regulatory regime, including the Private Native Forest legislation and proposed code of practice, nor the current market regime encourages sustainable management of the State's private timber resources. The Southern Cross Group has designed an effective and simple way of fostering and rewarding good stewardship of private native forests. Good stewardship may be viewed as a 'duty of care' responsibility that should be enforced by legislation, or as an environmental service that should be recognised and rewarded. The distinction is academic: the reality is that incentives are more effective than punitive regulations. Good environmental outcomes for most forests depend on active management and, especially in the case of private native forests, on incentives for continuing management. The challenge is to devise an equitable scheme that sends the right signals for forest management, is cost-effective to administer, and represents a worthwhile investment in terms of the public good generated. We believe the way forward is with simple, transparent indicators that provide an immediate and ongoing incentive. Under our proposal, landholders will receive an annual cash payment as a reward for progress towards specific outcomes. Rather than complicated targets, we are proposing a simple, two-tiered system that will give enough incentive to landholders to provide the environmental services desired by the community. The first tier rewards and encourages landholders to regenerate more forest, to retain big trees, and to stimulate tree growth on private land. The second tier rewards and encourages stewardship of endangered species and ecological communities. These incentives will be simple to apply and audit, and will encourage landholders to learn about and encourage biodiversity on their land, and to consider it part of their income portfolio. This system will contribute to farmers seeing forests as core business, both as part of their income stream and as part of their environmental stewardship responsibilities. When all farmers view forests in this way, Australia will reap the benefit of forests that are more diverse and productive, and a forest estate that no longer continues to shrink

Effects of diet forage source and neutral detergent fiber content on milk production of dairy cattle and methane emissions determined using GreenFeed and respiration chamber techniques

Strategies to mitigate greenhouse gas emissions from dairy cattle are unlikely to be adopted if production or profitability is reduced. The primary objective of this study was to examine the effects of high maize silage (MS) vs. high grass silage (GS) diets, without or with added neutral-detergent fiber (NDF) on milk production and methane emission of dairy cattle, using GreenFeed (GF) or respiration chamber (RC) techniques for methane emission measurements. Experiment 1 was 12-wks in duration with a randomized block continuous design and 40 Holstein cows (74 d in milk; DIM) in free-stall housing, assigned to 1 of 4 dietary treatments (n = 10 per treatment), according to calving date, parity and milk yield. Milk production and dry matter intake (DMI) were measured daily, and milk composition measured weekly, with methane yield (g/kg DMI) estimated using a GF unit (wks 10 to 12). Experiment 2 was a 4 × 4 Latin Square Design with 5-wk periods and 4 dairy cows (114 DIM) fed the same 4 dietary treatments as in experiment 1. Measurements of DMI, milk production and composition occurred in wk 4, and DMI, milk production and methane yield were measured for 2 d in RC during wk 5. Dietary treatments for both experiments were fed as TMRs offered ad libitum and containing 500 g silage/kg DM comprised of either 75:25 MS:GS (MS) or 25:75 MS:GS (GS), without or with added NDF from chopped straw and soy hulls (+47 g NDF/kg DM; MSNDF and GSNDF). In both experiments, compared to high GS, cows fed high MS had a higher (P = 0.01) DMI, greater (P = 0.01) milk production, and lower (P = 0.02) methane yield (24% lower in experiment 1 using GF and 8% lower in experiment 2 using RC). Added NDF increased (or tended to increase) methane yield for high MS, but not high GS diets (P = 0.02 for experiment 1 and P = 0.10 for experiment 2, forage type × NDF interaction). In the separate experiments the GF and RC methods detected similar dietary treatment effects on methane emission (expressed as g/d and g/kg DMI), although the magnitude of the difference varied between experiments for dietary treatments Overall methane emission and yield were 448 g/d and 20.9 g/kg DMI using GF for experiment 1 using GF and 458 g/d and 23.8 g/kg DMI for experiment 2 using RC, respectively

A mitochondrial-targeted cyclosporin A with high binding affinity for cyclophilin D yields improved cytoprotection of cardiomyocytes

Mitochondrial CyP-D (cyclophilin-D) catalyses formation of the PT (permeability transition) pore, a key lesion in the pathogenesis of I/R (ischaemia/reperfusion) injury. There is evidence [Malouitre, Dube, Selwood and Crompton (2010) Biochem. J. 425, 137–148] that cytoprotection by the CyP inhibitor CsA (cyclosporin A) is improved by selective targeting to mitochondria. To investigate this further, we have developed an improved mtCsA (mitochondrial-targeted CsA) by modifying the spacer linking the CsA to the TPP+ (triphenylphosphonium) (mitochondrial-targeting) cation. The new mtCsA exhibits an 18-fold increase in binding affinity for CyP-D over the prototype and a 12-fold increase in potency of inhibition of the PT in isolated mitochondria, owing to a marked decrease in non-specific binding. The cytoprotective capacity was assessed in isolated rat cardiomyocytes subjected to transient glucose and oxygen deprivation (pseudo-I/R). The new mtCsA was maximally effective at lower concentrations than CsA (3–15 nM compared with 50–100 nM) and yielded improved cytoprotection for up to 3 h following the pseudo-ischaemic insult (near complete compared with 40%). These data indicate the potential value of selective CyP-D inhibition in cytoprotection

Detection of circulating tumour DNA is associated with inferior outcomes in Ewing sarcoma and osteosarcoma: a report from the Children's Oncology Group.

BackgroundNew prognostic markers are needed to identify patients with Ewing sarcoma (EWS) and osteosarcoma unlikely to benefit from standard therapy. We describe the incidence and association with outcome of circulating tumour DNA (ctDNA) using next-generation sequencing (NGS) assays.MethodsA NGS hybrid capture assay and an ultra-low-pass whole-genome sequencing assay were used to detect ctDNA in banked plasma from patients with EWS and osteosarcoma, respectively. Patients were coded as positive or negative for ctDNA and tested for association with clinical features and outcome.ResultsThe analytic cohort included 94 patients with EWS (82% from initial diagnosis) and 72 patients with primary localised osteosarcoma (100% from initial diagnosis). ctDNA was detectable in 53% and 57% of newly diagnosed patients with EWS and osteosarcoma, respectively. Among patients with newly diagnosed localised EWS, detectable ctDNA was associated with inferior 3-year event-free survival (48.6% vs. 82.1%; p = 0.006) and overall survival (79.8% vs. 92.6%; p = 0.01). In both EWS and osteosarcoma, risk of event and death increased with ctDNA levels.ConclusionsNGS assays agnostic of primary tumour sequencing results detect ctDNA in half of the plasma samples from patients with newly diagnosed EWS and osteosarcoma. Detectable ctDNA is associated with inferior outcomes