Polymer nanocarrier system for endosome escape and timed release of siRNA with complete gene silencing and cell death in cancer cells

An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U-2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA.

Does the fair basing "problem child" escape Lockwood?

In an earlier article the concept of fair basing in Australian patent law was described as a "problem child", often unruly and unpredictable in practice, but nevertheless understandable and useful in policy terms. The article traced the development of several different branches of patent law that were swept under the nomenclature of "fair basing" in Britain in 1949. It then went on to examine the adoption of fair basis into Australian law, the modern interpretation of the requirement, and its problems. This article provides an update. After briefly recapping on the relevant historical issues, it examines the recent Lockwood "internal" fair basing case in the Federal and High Courts.

Syntaxin 11 binds Vti1b and regulates late endosome to lysosome fusion in macrophages

Syntaxin 11 (Stx11) is a SNARE protein enriched in cells of the immune system. Loss or mutation of Stx11 results in familial hemophagocytic lymphohistiocytosis type-4 (FHL-4), an autosomal recessive disorder of immune dysregulation characterized by high levels of inflammatory cytokines along with defects in T-cell and natural killer cell function. We show here Stx11 is located on endosomalmembranes including late endosomes and lysosomes in macrophages. While Stx11 did not form a typical trans-SNARE complex, it did bind to the Q-SNARE Vti1b and was able to regulate the availability of Vti1b to form the Q-SNARE complexes Stx6/Stx7/Vtib and Stx7/Stx8/Vti1b. The mutant form of Stx11 sequestered Vti1b from forming the Q-SNARE complex that mediates late endosome to lysosome fusion. Depletion of Stx11 in activated macrophages leads to an accumulation of enlarged late endocytic compartments, increased trafficking to the cell surface and inhibition of late endosome to lysosome fusion. These phenotypes arerescued by the expression of an siRNA-resistant Stx11 construct in Stx11-depleted cells. Our results suggest that by regulating the availability of Vti1b, Stx11 regulates trafficking steps between late endosomes, lysosomes and the cell surface in macrophages.

The role of the recycling endosome in regulating lamellipodia formation and macrophage migration

Cell migration is a highly complex process that requires the extension of cell membrane in the direction of travel. This membrane is continuously remodeled to expand the leading edge and alter its membrane properties. For a long time it has been known that there is a continual flow of polarized membrane traffic towards the leading edge during migration and that this trafficking is essential for cell migration. However, there is little information on how the cell coordinates exocytosis at the leading edge. It is also unclear whether these internal membranes are incorporated into the leading edge or are just delivering the necessary proteins for migration to occur. We have shown that recycling endosome membrane is incorporated into the plasma membrane at the leading edge to expand the membrane and at the same time delivers receptors to the leading edge to mediate migration. In order for this to happen the surface Q-SNARE complex Stx4/SNAP23 translocates to the leading edge where it binds to the R-SNARE VAMP3 on the recycling endosome allowing incorporation into the plasma membrane. Loss of any one of the components of this complex reduces efficient lamellipodia formation and restrains cell migration.

Recycling endosome membrane incorporation into the leading edge regulates lamellipodia formation and macrophage migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R-SNARE VAMP3 on the recycling endosome partnering with the surface Q-SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3-mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3-mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.

Flightless, secreted through a late endosome/lysosome pathway, binds LPS and dampens cytokine secretion.

Flightless (Flii) is upregulated in response to wounding and has been shown to function in wound closure and scarring. In macrophages intracellular Flii negatively modulates TLR signalling and dampens cytokine production. We now show that Flii is constitutively secreted from macrophages and fibroblasts and is present in human plasma. Secretion from fibroblasts is upregulated in response to scratch wounding and LPS-activated macrophages also temporally upregulate their secretion of Flii. Using siRNA, wild-type and mutant proteins we show that Flii is secreted via a late endosomal/lysosomal pathway that is regulated by Rab7 and Stx11. Flii contains 11 leucine rich repeat (LRR) domains in its N-terminus that have nearly 50% similarity to those in the extracellular pathogen binding portion of Toll-like receptor 4 (TLR4). We show secreted Flii can also bind LPS and has the ability to alter macrophage activation. LPS activation of macrophages in Flii depleted conditioned media leads to enhanced macrophage activation and increased TNF secretion compared to cells activated in the presence of Flii. These results show secreted Flii binds to LPS and in doing so alters macrophage activation and cytokine secretion, suggesting that like the intracellular pool of Flii, secreted Flii also has the ability to alter inflammation.

An influenza virus inspired polymer system for the timed release of siRNA

Small interfering RNA silences specific genes by interfering with mRNA translation, and acts to modulate or inhibit specific biological pathways; a therapy that holds great promise in the cure of many diseases. However, the naked small interfering RNA is susceptible to degradation by plasma and tissue nucleases and due to its negative charge unable to cross the cell membrane. Here we report a new polymer carrier designed to mimic the influenza virus escape mechanism from the endosome, followed by a timed release of the small interfering RNA in the cytosol through a self-catalyzed polymer degradation process. Our polymer changes to a negatively charged and non-toxic polymer after the release of small interfering RNA, presenting potential for multiple repeat doses and long-term treatment of diseases.

Escape from apoptosis after prolonged serum deprivation is associated with the regulation of the mitochondrial death pathway by Bcl-x(l)

Bcl-x(l) and Bax play important roles in the regulation of apoptosis. This study investigated the involvement of the mitochondrial death pathway and the role of Bcl-x(l) and Bax in the escape from apoptosis after prolonged serum deprivation in Madin-Darby canine kidney (MDCK) cells. Low level apoptosis and basal activity of the mitochondrial death pathway were detectable in normal cell growth. In serum deprivation, mitosis was partially suppressed, and the mitochondrial activity was stimulated. The level of apoptosis continuously rose over 48 h. This rise was concomitant with the increasing presence of cytochrome c in cytosol. However, both apoptosis and cytosolic cytochrome c fell dramatically at 72 h. Elevation of whole cell Bcl-x(l) and redistribution of Bcl-x(l) protein from cytosol to the membrane at 48 h and 72 h was observed. Redistribution of Bax protein from the membrane to cytosol occurred at 24 h, and remained steady to 72 h. Bax/Bcl-x(l) coimmunoprecipitation by anti-Bax antibody showed reduced Bax/Bcl-x(l) interaction at the membrane at 72 h, but not at 24 or 48 h. These results suggest that apoptosis upon serum withdrawal results from the leakage of cytochrome c to cytosol. Amelioration of the leakage of cytochrome c and apoptosis requires not only the increase of Bcl-x(l)/Bax ratio, but also the release of Bcl-x(l) from Bax at the membrane.

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

The design and development of music eScape: A new iPhone mood management music App

The physical, emotional, educational and social developmental challenges of adolescence can be associated with high levels of emotional vulnerability. Thus, the development of effective emotion-regulation strategies is crucial during this time period. Young people commonly use music to identify, express and regulate their emotions. Modern mobile technology provides an engaging, easily accessible means of assisting young people through music. A systematic contextual review identified 20 iPhone applications addressing emotions through music and two independent raters, using the Mobile App Rating Scale (MARS), evaluated the quality of the apps. Their characteristics, key features and overall quality will be presented. Three participatory design workshops (N=13, 6 males, 7 females; age 15-25) were conducted to explore young people’s use of music to enhance wellbeing. Young people were also asked to trial existing mood and music apps and to conceptualise their ultimate mood targeting music application. A thematic analysis of the participatory design workshops content identified the following music affect-regulation strategies: relationship building, modifying cognitions, modifying emotions, and immersing in emotions. The application of the key learnings from the mobile app review and participatory design workshops and the design and development of the music eScape app were presented and implications for future research was discussed.

'Music eScape': mHealth application

Research Background Young people’s avid use of mobile technologies in daily life has led to an increase in the design and research on mHealth (mobile health) interventions targeting young people. ‘Music eScape’ is a mobile based mood regulation app that uses an innovative approach to promoting young people’s wellbeing using music. Research Question The design, research, development and evaluation of ‘Music eScape’ addressed a number of research questions from across the fields of Psychology and Interactive and Visual Design. The specific design research question addressed was: How can interaction and visual design be utilized to promote and enable young people to effectively regulate their mood using music and how can the new design further promote their experience of empowerment, control and agency over actively directing their mood journey? Research Contribution Innovation and New Knowledge Through its unique visual interface design and interactivity, the application presents a novel approach to promoting young people’s wellbeing using music and a specific function that allows users to ‘draw’ their mood journey in order to generate a playlist. The mobile app is the first to contain a function that enables users to plan their mood journey and exercise a sense of agency, intentional choice and control over the mood shift and by extension, their wellbeing. The feature ‘drawing’ interface was designed by Oksana Zelenko using participatory design research and Russell’s circumplex model of affect (1980) to inform the key visual design concept and underpinning interaction design. Research Significance The significance of the design research component within the larger interdisciplinary practices that have informed ‘Music eScape’ (e.g. field of psychology, reported through journal articles and other related outcomes), is the unique visual and interactive presentation of participant data and music therapy research within the app interface and interaction design which improves and increases young people’s engagement with the health messages it contains. The industry quality standard is further demonstrated by the launch on Apple iTunes. This demonstrates the application meets the high professional requirements for national release and meets international standards. The app also creates a new benchmark for the quality of health apps on the market as it marks the industry release of a trialled evidence-based mHealth intervention co-designed with young people.

Post-transcriptional regulation in the nucleus and cytoplasm: study of mean time to threshold (MTT) and narrow escape problem

Messenger RNAs (mRNAs) can be repressed and degraded by small non-coding RNA molecules. In this paper, we formulate a coarsegrained Markov-chain description of the post-transcriptional regulation of mRNAs by either small interfering RNAs (siRNAs) or microRNAs (miRNAs). We calculate the probability of an mRNA escaping from its domain before it is repressed by siRNAs/miRNAs via cal- culation of the mean time to threshold: when the number of bound siRNAs/miRNAs exceeds a certain threshold value, the mRNA is irreversibly repressed. In some cases,the analysis can be reduced to counting certain paths in a reduced Markov model. We obtain explicit expressions when the small RNA bind irreversibly to the mRNA and we also discuss the reversible binding case. We apply our models to the study of RNA interference in the nucleus, examining the probability of mRNAs escaping via small nuclear pores before being degraded by siRNAs. Using the same modelling framework, we further investigate the effect of small, decoy RNAs (decoys) on the process of post-transcriptional regulation, by studying regulation of the tumor suppressor gene, PTEN : decoys are able to block binding sites on PTEN mRNAs, thereby educing the number of sites available to siRNAs/miRNAs and helping to protect it from repression. We calculate the probability of a cytoplasmic PTEN mRNA translocating to the endoplasmic reticulum before being repressed by miRNAs. We support our results with stochastic simulations

Intimate Drowning

intimate drowning 50 minute performance + installation work ice | salt | tears | This work is about death. Grief The relationships before The aftermath - of confusion, violence, isolation The never ending questions The devastating loss and paranoia "Since my wife died, I have spent the last six years treading water - trying to stop myself from drowning. Sometimes I catch myself not breathing. I have to remind myself that I can't live underwater no matter how much I want to." Grief. Loss. Tears. Fear. Sadness Water. Milk. Salt. Ice Falling. Waiting Submerged. Suffocated. Broken ties Intention. Lack of focus. Intensity of focus Fighting. Screaming. Wailing Blue. White. Black. Blackness The doors open: we walk through a gauze curtain and discover a dark space with a square of light in the middle of the room. As we walk closer to the light, we see a girl writing in charcoal on the floor around a square box filled with milk. She is writing the same thing over and over. The more she writes the more desperate she becomes: I am listening… We have to keep walking past. She isn’t writing for us. We find our seats Two people: one slowly breaking the hundreds of fragile strings that tie her to the other. The other is pleading with her to stop: Please. Please don’t. Please Avril. …Please don’t One girl facing away from us. She is slowly swimming on the spot without water. Projected next to her are images of her drowning under water. Salt falls in front of her. Behind her. A wall of salt. She is bound to the spot. Underwater and still breathing. Swimming in her own tears. She won’t escape. She wants to stay, but desires nothing Two people standing in a large square box filled with milk. They start in intimacy. The relationship begins to dissolve before us. One fights to be with/on/behind the other. The other fights her off. The milk is splashed. Why aren't they being careful? In the darkness there is scrubbing. Someone is scrubbing the floor. The other girl is on her knees trying to erase the original writing. The traces left behind that we have no control over. We only see her for a second, but hear her in the darkness. Scrubbing. It is pointless. You can't erase the past.