Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein

Evaluation of the efficiency of tumor and tissue delivery of carrier-mediated agents (CMA) and small molecule (SM) agents in mice using a novel pharmacokinetic (PK) metric: relative distribution index over time (RDI-OT)

The pharmacokinetics (PK) of carrier-mediated agents (CMA) is dependent upon the carrier system. As a result, CMA PK differs greatly from the PK of small molecule (SM) drugs. Advantages of CMAs over SMs include prolonged circulation time in plasma, increased delivery to tumors, increased antitumor response, and decreased toxicity. In theory, CMAs provide greater tumor drug delivery than SMs due to their prolonged plasma circulation time. We sought to create a novel PK metric to evaluate the efficiency of tumor and tissue delivery of CMAs and SMs. We conducted a study evaluating the plasma, tumor, liver, and spleen PK of CMAs and SMs in mice bearing subcutaneous flank tumors using standard PK parameters and a novel PK metric entitled relative distribution over time (RDI-OT), which measures efficiency of delivery. RDI-OT is defined as the ratio of tissue drug concentration to plasma drug concentration at each time point. The standard concentration versus time area under the curve values (AUC) of CMAs were higher in all tissues and plasma compared with SMs. However, 8 of 17 SMs had greater tumor RDI-OT AUC0–last values than their CMA comparators and all SMs had greater tumor RDI-OT AUC0–6 h values than their CMA comparators. Our results indicate that in mice bearing flank tumor xenografts, SMs distribute into tumor more efficiently than CMAs. Further research in additional tumor models that may more closely resemble tumors seen in patients is needed to determine if our results are consistent in different model systems

MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer

Background: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. Methods: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. Results: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. Conclusion: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype

Crop Updates 2001 - Lupins

This session covers twenty six papers from different authors: INTRODUCTION, 1. Introduction, Dr Mark Sweetingham LUPIN RESEARCH AND INDUSTRY DEVELOPMENT, Agriculture Western Australia VARIETIES 2. Lupin variety performance: Are you making the most of it? Bevan J. Buirchell, Agriculture Western Australia 3. Adaption of restricted-branching lupins in Western Australia, Bob French and Laurie Wahlsten, Agriculture Western Australia 4. Isolated microspore culture of lupin for production of doubled haploids, Dr Janet Wroth, Dr Kirsty Bayliss and A/Prof. Wallace Cowling, Plant Sciences, Faculty of Agriculture, The University of Western Australia NUTRITION 5. Banding manganese fertiliser below the seed increases seed yields of narrow-leafed lupins, R.F. Brennan, Agriculture Western Australia 6. Residual value of manganese fertiliser for lupin grain production, R.F. Brennan, Agriculture Western Australia AGRONOMY 7. Lupin seeding density, Miles Dracup, Agriculture Western Australia, Nick Galwey, University of Western Australia and Bob Thomson, University of Western AustraliaPESTS AND DISEASES 8. Anthracnose in lupins – understanding the risk, Moin Salam, Art Diggle, Geoff Thomas, Mark Sweetinghamand Bill O’Neill, Agriculture Western Australia 9. Implications of the ‘green bridge’ for viral and fungal disease carry-over between seasons, Debbie Thackray, Agriculture Western Australia and Centre for Legumes in Mediterranean Agriculture 10. Insect pest development in WA via the ‘green bridge’, Kevin Walden, Agriculture Western Australia 11. Lupin anthracnose – seed infection thresholds, Geoff Thomas, Agriculture Western Australia 12. Identification and characterisation of resistance genes to Phomopsis blight in narrow-leafed lupin, M. Shankar1, M.W. Sweetingham1&2 and W.A. Cowling1&3 , 1Co-operative Research Centre for Legumes in Mediterranean Agriculture, 2Agriculture Western Australia, 3Plant Sciences 13. Plant disease diagnostics, Dominie Wright and Nichole Burges, Agriculture Western Australia 14. Detection of strains of Phomopsis exhibiting species preference in lupins, M. Shankar, 1Co-operative Research Centre for Legumes in Mediterranean Agriculture and M.W. Sweetingham, Agriculture Western Australia 15. Potential alternate host for the lupin anthracnose pathogen, Geoff Thomasa, Hu’aan Yangb, Mark Sweetinghamab and Ming Pei Youa, aAgriculture Western Australia, bCooperative Research Centre for Legumes in Mediterranean Agriculture WEEDS 16. Wild radish – the implications for our rotations, Dr David Bowran, Centre for Cropping Systems 17. Competitiveness of wild radish in a wheat – lupin rotation, Abul Hashem, Nerys Wilkins, and Terry Piper, Agriculture Western Australia 18. Population explosion and persistence of wild radish in a wheat-lupin rotation, Abul Hashem, Nerys Wilkins, Aik Cheam and Terry Piper, Agriculture Western Australia 19. Inter-row knockdowns for profitable lupins, Paul Blackwell, Agriculture Western Australia, Miles Obst, Mingenew 20. Is it safe to use 2,4-D Ester 80% pre-sowing when furrow sowing lupins? Andrew Sandison, Elders Ltd QUALITY AND MARKET DEVELOPMENT 21. Lupin protein – what we know, Bill O’Neill, Agriculture Western Australia 22. Foliar N application increases grain protein in lupins, Bob French and Laurie Wahlsten, Agriculture Western Australia 23. Can lupin grain protein be increased with Flexi-N? Cameron Weeks, Erin Hasson, Mingenew-Irwin Group and Luigi Moreschi, CSBP futurefarm 24. Putting a value on lupin use in the aquaculture industry: a fishy business? Brett D. Glencross, Fisheries WA, Fremantle Maritime Centre, Fremantle 25. Selection for thinner seed coats and pod walls in lupins, Jon Clements, Centre for Legumes in Mediterranean Agriculture and Miles Dracup, Agriculture Western Australia 26. Assessing the nutritional benefit of Australian sweet lupin (Lupinus angustifolius) in human foods, Ramon Hall (SPIRT PhD scholar), Stuart Johnson, Madeleine Ball, Deakin University, Melbourne, Sofia Sipsas and David Petterson, Agriculture Western Australi

Understanding the influences on successful quality improvement in emergency general surgery: learning from the RCS Chole-QuIC project

Abstract: Background: Acute gallstone disease is the highest volume Emergency General Surgical presentation in the UK. Recent data indicate wide variations in the quality of care provided across the country, with national guidance for care delivery not implemented in most UK hospitals. Against this backdrop, the Royal College of Surgeons of England set up a 13-hospital quality improvement collaborative (Chole-QuIC) to support clinical teams to reduce time to surgery for patients with acute gallstone disease requiring emergency cholecystectomy. Methods: Prospective, mixed-methods process evaluation to answer the following: (1) how was the collaborative delivered by the faculty and received, understood and enacted by the participants; (2) what influenced teams’ ability to improve care for patients requiring emergency cholecystectomy? We collected and analysed a range of data including field notes, ethnographic observations of meetings, and project documentation. Analysis was based on the framework approach, informed by Normalisation Process Theory, and involved the creation of comparative case studies based on hospital performance during the project. Results: Chole-QuIC was delivered as planned and was well received and understood by participants. Four hospitals were identified as highly successful, based upon a substantial increase in the number of patients having surgery in line with national guidance. Conversely, four hospitals were identified as challenged, achieving no significant improvement. The comparative analysis indicate that six inter-related influences appeared most associated with improvement: (1) achieving clarity of purpose amongst site leads and key stakeholders; (2) capacity to lead and effective project support; (3) ideas to action; (4) learning from own and others’ experience; (5) creating additional capacity to do emergency cholecystectomies; and (6) coordinating/managing the patient pathway. Conclusion: Collaborative-based quality improvement is a viable strategy for emergency surgery but success requires the deployment of effective clinical strategies in conjunction with improvement strategies. In particular, achieving clarity of purpose about proposed changes amongst key stakeholders was a vital precursor to improvement, enabling the creation of additional surgical capacity and new pathways to be implemented effectively. Protected time, testing ideas, and the ability to learn quickly from data and experience were associated with greater impact within this cohort

Lessons learned: Symbiotic autonomous robot ecosystem for nuclear environments

Nuclear facilities have a regulatory requirement to measure radiation levels within Post Operational Cleanout (POCO) around nuclear facilities each year, resulting in a trend towards robotic deployments to gain an improved understanding during nuclear decommissioning phases. The UK Nuclear Decommissioning Authority supports the view that human-in-the-loop robotic deployments are a solution to improve procedures and reduce risks within radiation haracterisation of nuclear sites. We present a novel implementation of a Cyber-Physical System (CPS) deployed in an analogue nuclear environment, comprised of a multi-robot team coordinated by a human-in-the-loop operator through a digital twin interface. The development of the CPS created efficient partnerships across systems including robots, digital systems and human. This was presented as a multi-staged mission within an inspection scenario for the heterogeneous Symbiotic Multi-Robot Fleet (SMuRF). Symbiotic interactions were achieved across the SMuRF where robots utilised automated collaborative governance to work together where a single robot would face challenges in full characterisation of radiation. Key contributions include the demonstration of symbiotic autonomy and query-based learning of an autonomous mission supporting scalable autonomy and autonomy as a service. The coordination of the CPS was a success and displayed further challenges and improvements related to future multi-robot fleets

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Intracellular protein determination using droplet-based immunoassays

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from 50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells

Clinicopathological parameters, recurrence, locoregional and distant metastasis in 115 T1-T2 oral squamous cell carcinoma patients

The incidence of oral squamous cell carcinoma remains high. Oral and oro-pharyngeal carcinomas are the sixth most common cancer in the world. Several clinicopathological parameters have been implicated in prognosis, recurrence and survival, following oral squamous cell carcinoma. In this retrospective analysis, clinicopathological parameters of 115 T1/T2 OSCC were studied and compared to recurrence and death from tumour-related causes. The study protocol was approved by the Joint UCL/UCLH committees of the ethics for human research. The patients' data was entered onto proformas, which were validated and checked by interval sampling. The fields included a range of clinical, operative and histopathological variables related to the status of the surgical margins. Data collection also included recurrence, cause of death, date of death and last clinic review. Causes of death were collated in 4 categories (1) death from locoregional spread, (2) death from distant metastasis, (3) death from bronchopulmonary pneumonia, and (4) death from any non-tumour event that lead to cardiorespiratory failure. The patients' population comprised 65 males and 50 females. Their mean age at the 1 diagnosis of OSCC was 61.7 years. Two-thirds of the patients were Caucasians. Primary sites were mainly identified in the tongue, floor of mouth (FOM), buccal mucosa and alveolus. Most of the identified OSCCs were low-risk (T1N0 and T2N0). All patients underwent primary resection neck dissection and reconstruction when necessary. Twenty-two patients needed adjuvant radiotherapy. Pathological analysis revealed that half of the patients had moderately differentiated OSCC. pTNM slightly differed from the cTNM and showed that 70.4% of the patients had low-risk OSCC. Tumour clearance was ultimately achieved in 107 patients. Follow-up resulted in a 3-year survival of 74.8% and a 5-year survival of 72.2%. Recurrence was identified in 23 males and 20 females. The mean age of 1 diagnosis of the recurrence group was 59.53 years. Most common oral sites included the lateral border of tongue and floor of mouth. Recurrence was associated with clinical N-stage disease. The surgical margins in this group was evaluated and found that 17 had non-cohesive invasion, 30 had dysplasia at margin, 21 had vascular invasion, 9 had nerve invasion and 3 had bony invasion. Severe dysplasia was present in 37 patients. Tumour clearance was achieved in only 8 patients. The mean depth of tumour invasion in the recurrence group was 7.6 mm. An interesting finding was that 5/11 patients who died of distant metastasis had their primary disease in the tongue. Nodal disease comparison showed that 8/10 patients who died of locoregional metastasis and 8/11 patients who died from distant metastasis had clinical nodal involvement. Comparing this to pathological nodal disease (pTNM) showed that 10/10 patients and 10/11 patients who died from locoregional and distant metastasis, respectively, had nodal disease. All patients who died from locoregional and distant metastasis were shown to have recurrence after the primary tumour resection. Squamous cell carcinoma of the oral cavity has a poor overall prognosis with a high tendency to recur at the primary site and extend to involve the cervical lymph nodes. Several clinicopathological parameters can be employed to assess outcome, recurrence and overall survival. © 2010 Jerjes et al; licensee BioMed Central Ltd