High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50 nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics.

Correlating liposomal adjuvant characteristics to in-vivo cell-mediated immunity using a novel Mycobacterium tuberculosis fusion protein:a multivariate analysis study

Objective In this study, we have used a chemometrics-based method to correlate key liposomal adjuvant attributes with in-vivo immune responses based on multivariate analysis. Methods The liposomal adjuvant composed of the cationic lipid dimethyldioctadecylammonium bromide (DDA) and trehalose 6,6-dibehenate (TDB) was modified with 1,2-distearoyl-sn-glycero-3-phosphocholine at a range of mol% ratios, and the main liposomal characteristics (liposome size and zeta potential) was measured along with their immunological performance as an adjuvant for the novel, postexposure fusion tuberculosis vaccine, Ag85B-ESAT-6-Rv2660c (H56 vaccine). Partial least square regression analysis was applied to correlate and cluster liposomal adjuvants particle characteristics with in-vivo derived immunological performances (IgG, IgG1, IgG2b, spleen proliferation, IL-2, IL-5, IL-6, IL-10, IFN-γ). Key findings While a range of factors varied in the formulations, decreasing the 1,2-distearoyl-sn-glycero-3-phosphocholine content (and subsequent zeta potential) together built the strongest variables in the model. Enhanced DDA and TDB content (and subsequent zeta potential) stimulated a response skewed towards a cell mediated immunity, with the model identifying correlations with IFN-γ, IL-2 and IL-6. Conclusion This study demonstrates the application of chemometrics-based correlations and clustering, which can inform liposomal adjuvant design.

Microfluidic-controlled manufacture of liposomes for the solubilisation of a poorly water soluble drug

Besides their well-described use as delivery systems for water-soluble drugs, liposomes have the ability to act as a solubilizing agent for drugs with low aqueous solubility. However, a key limitation in exploiting liposome technology is the availability of scalable, low-cost production methods for the preparation of liposomes. Here we describe a new method, using microfluidics, to prepare liposomal solubilising systems which can incorporate low solubility drugs (in this case propofol). The setup, based on a chaotic advection micromixer, showed high drug loading (41 mol%) of propofol as well as the ability to manufacture vesicles with at prescribed sizes (between 50 and 450 nm) in a high-throughput setting. Our results demonstrate the ability of merging liposome manufacturing and drug encapsulation in a single process step, leading to an overall reduced process time. These studies emphasise the flexibility and ease of applying lab-on-a-chip microfluidics for the solubilisation of poorly water-soluble drugs.

Designing liposomal adjuvants for the next generation of vaccines

Liposomes not only offer the ability to enhance drug delivery, but can effectively act as vaccine delivery systems and adjuvants. Their flexibility in size, charge, bilayer rigidity and composition allow for targeted antigen delivery via a range of administration routes. In the development of liposomal adjuvants, the type of immune response promoted has been linked to their physico-chemical characteristics, with the size and charge of the liposomal particles impacting on liposome biodistribution, exposure in the lymph nodes and recruitment of the innate immune system. The addition of immunostimulatory agents can further potentiate their immunogenic properties. Here, we outline the attributes that should be considered in the design and manufacture of liposomal adjuvants for the delivery of sub-unit and nucleic acid based vaccines.

Dynamic variability in adaptive systems

One of the reasons for using variability in the software product line (SPL) approach (see Apel et al., 2006; Figueiredo et al., 2008; Kastner et al., 2007; Mezini & Ostermann, 2004) is to delay a design decision (Svahnberg et al., 2005). Instead of deciding on what system to develop in advance, with the SPL approach a set of components and a reference architecture are specified and implemented (during domain engineering, see Czarnecki & Eisenecker, 2000) out of which individual systems are composed at a later stage (during application engineering, see Czarnecki & Eisenecker, 2000). By postponing the design decisions in such a manner, it is possible to better fit the resultant system in its intended environment, for instance, to allow selection of the system interaction mode to be made after the customers have purchased particular hardware, such as a PDA vs. a laptop. Such variability is expressed through variation points which are locations in a software-based system where choices are available for defining a specific instance of a system (Svahnberg et al., 2005). Until recently it had sufficed to postpone committing to a specific system instance till before the system runtime. However, in the recent years the use and expectations of software systems in human society has undergone significant changes.Today's software systems need to be always available, highly interactive, and able to continuously adapt according to the varying environment conditions, user characteristics and characteristics of other systems that interact with them. Such systems, called adaptive systems, are expected to be long-lived and able to undertake adaptations with little or no human intervention (Cheng et al., 2009). Therefore, the variability now needs to be present also at system runtime, which leads to the emergence of a new type of system: adaptive systems with dynamic variability.

Nanotechnology and microfluidics:formulation design and on-chip manufacture of nanoparticles

Nanoparticles offer an ideal platform for the delivery of small molecule drugs, subunit vaccines and genetic constructs. Besides the necessity of a homogenous size distribution, defined loading efficiencies and reasonable production and development costs, one of the major bottlenecks in translating nanoparticles into clinical application is the need for rapid, robust and reproducible development techniques. Within this thesis, microfluidic methods were investigated for the manufacturing, drug or protein loading and purification of pharmaceutically relevant nanoparticles. Initially, methods to prepare small liposomes were evaluated and compared to a microfluidics-directed nanoprecipitation method. To support the implementation of statistical process control, design of experiment models aided the process robustness and validation for the methods investigated and gave an initial overview of the size ranges obtainable in each method whilst evaluating advantages and disadvantages of each method. The lab-on-a-chip system resulted in a high-throughput vesicle manufacturing, enabling a rapid process and a high degree of process control. To further investigate this method, cationic low transition temperature lipids, cationic bola-amphiphiles with delocalized charge centers, neutral lipids and polymers were used in the microfluidics-directed nanoprecipitation method to formulate vesicles. Whereas the total flow rate (TFR) and the ratio of solvent to aqueous stream (flow rate ratio, FRR) was shown to be influential for controlling the vesicle size in high transition temperature lipids, the factor FRR was found the most influential factor controlling the size of vesicles consisting of low transition temperature lipids and polymer-based nanoparticles. The biological activity of the resulting constructs was confirmed by an invitro transfection of pDNA constructs using cationic nanoprecipitated vesicles. Design of experiments and multivariate data analysis revealed the mathematical relationship and significance of the factors TFR and FRR in the microfluidics process to the liposome size, polydispersity and transfection efficiency. Multivariate tools were used to cluster and predict specific in-vivo immune responses dependent on key liposome adjuvant characteristics upon delivery a tuberculosis antigen in a vaccine candidate. The addition of a low solubility model drug (propofol) in the nanoprecipitation method resulted in a significantly higher solubilisation of the drug within the liposomal bilayer, compared to the control method. The microfluidics method underwent scale-up work by increasing the channel diameter and parallelisation of the mixers in a planar way, resulting in an overall 40-fold increase in throughput. Furthermore, microfluidic tools were developed based on a microfluidics-directed tangential flow filtration, which allowed for a continuous manufacturing, purification and concentration of liposomal drug products.

Global cropland and greenhouse gas impacts of UK food supply are increasingly located overseas

Date of Acceptance: 04/12/2016 © 2016 The Author(s). This work was supported by a University of Aberdeen Environment and Food Security Theme/the James Hutton Institute PhD studentship, and contributes to the Scottish Food Security Alliance-Crops and the Belmont Forum supported DEVIL project (NERC fund UK contribution: NE/M021327/1). J.M. and R.B.M. acknowledge funding from the Rural and Environment Science and Analytical Services, Scottish Government. T.K. acknowledges funding from the European Research Council Grant ERC-263522 (LUISE).

Rapid quantification and validation of lipid concentrations within liposomes

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and D-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

Control of the innate immune response by the mevalonate pathway.

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1β that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.

Understandings of transnational families in debates on Migration and development

International migration sets in motion a range of significant transnational processes that connect countries and people. How migration interacts with development and how policies might promote and enhance such interactions have, since the turn of the millennium, gained attention on the international agenda. The recognition that transnational practices connect migrants and their families across sending and receiving societies forms part of this debate. The ways in which policy debate employs and understands transnational family ties nevertheless remain underexplored. This article sets out to discern the understandings of the family in two (often intermingled) debates concerned with transnational interactions: The largely state and policydriven discourse on the potential benefits of migration on economic development, and the largely academic transnational family literature focusing on issues of care and the micro-politics of gender and generation. Emphasizing the relation between diverse migration-development dynamics and specific family positions, we ask whether an analytical point of departure in respective transnational motherhood, fatherhood or childhood is linked to emphasizing certain outcomes. We conclude by sketching important strands of inclusions and exclusions of family matters in policy discourse and suggest ways to better integrate a transnational family perspective in global migration-development policy.

Qualitätssicherung zwischen Diversifizierung der Hochschulen und Vereinheitlichung von Standards. Beiträge zur 2. AQ Austria Jahrestagung 2014

Die Jahrestagung 2014 der AQ Austria widmete sich einerseits dem Spannungsfeld zunehmender Diversifizierung von Hochschulen und standardisierten Qualitätssicherungsverfahren andererseits. Im vorliegenden Jahrestagungsband finden sich neben dem Hauptvortrag des Basler Universitätsrektors Loprieno über die Herausforderungen der Diversifizierung für Qualität sowie der Qualitätskultur an Hochschulen auch internationale Beiträge als Antwort aus der Sicht von Qualitätssicherungsagenturen auf die Diversifizierung. Weitere Artikel zu Themen wie duale Studiengänge, Studierbarkeit, Berufungsverfahren sowie Forschungskultur und Qualitätskultur runden die Diskussion über Qualitätssicherung und Diversifizierung in der vorliegenden Publikation ab. Mit Beiträgen von: Loprieno, Antonio; Hanft, Anke; Pichl, Elmar; Jackson, Stephen; Lund, Øystein; Cox, Jeremy; Fink, Kerstin; Brandstätter, Ursula; Bischof, Horst; Gaberscik, Gerald; Janger, Jürgen; Kastner, Johann; Steiger, Anna; Wilhelm, Elena; Holzinger, Helmut; Esca-Scheuringer, Heidi; Kecht, Maria-Regina; Schulmeister, Rolf; Haas, Johannes.

L’intégration, dans la communauté, des personnes présentant une déficience intellectuelle : étude des manifestations d'opposition ou d'appui du voisinage

La volonté d'intégrer, dans la communauté, les personnes présentant une déficience intellectuelle et de changer le mode de vie institutionnel par un mode de vie similaire à celui des autres citoyens, oriente depuis plusieurs années les services de réadaptation. Comme le souligne Kebbon (1987), ce désir d'intégrer les personnes présentant une déficience intellectuelle est largement issu du principe de la normalisation. D'abord formulé en Scandinavie par Nirje (1969) puis repris aux États-Unis par Wolfensberger (1972), ce principe statue qu'un effort constant doit être fait pour rendre normales les conditions de vie des personnes présentant une déficience intellectuelle. L'intégration est "une condition nécessaire à la normalisation et est habituellement le moyen d'y aboutir" (Kebbon, 1987; 69). Toutefois, selon plusieurs auteurs, le succès ou l'échec de l'intégration repose largement sur l'acceptation et l'appui du public (Eisenring et Pasche, 1981; lonescu, 1987; Kastner, Repucci et Pezzoli, 1979; Roth et Smith, 1983; Sandler et Robinson, 1981; Seltzer, 1984; Sternlicht, 1976). La connaissance des réactions des personnes qui vivent dans le voisinage d'une ressource d'hébergement pour personnes ayant une déficience intellectuelle s'avère donc essentielle afin de bien évaluer le processus d'intégration et d'offrir aux personnes déficientes une meilleure qualité de vie. Le mouvement de désinstitutionnalisation et d'intégration des personnes déficientes a été amorcé au Québec depuis une dizaine d'années. Or, on ne sait encore que peu de choses quant aux réactions de la population à cette intégration communautaire. Deux études québécoises (Coté, Ouellet et Lachance, 1990; lonescu et Despins, 1990) portant sur les attitudes envers l'intégration communautaire des personnes ayant une déficience intellectuelle apportent quelques renseignements. L'étude de lonescu et Despins réalisée auprès d'étudiants et d'étudiantes de niveau collégial et universitaire, montre que 85,5% des répondants sont favorables à l'intégration communautaire des personnes présentant une déficience intellectuelle. Celle de Côté et al. menée auprès du grand public, indique que les deux tiers des répondants ne seraient pas défavorables à la présence de ces personnes dans leur quartier. Toutefois, aucune étude québécoise réalisée auprès de personnes qui vivent directement en contact avec le phénomène de l'intégration des personnes présentant une déficience intellectuelle n'a été recensée. Comment réagissent les gens lorsqu'ils sont confrontés au phénomène de l'intégration? Acceptent-ils la présence des personnes ayant une déficience intellectuelle ou, au contraire, s'y opposent-ils? Comment manifestent-ils leur acceptation ou leur opposition? Voilà autant de questions auxquelles ce mémoire tentera de répondre.

HOLDING HANDS WHILE PARTING WAYS: EXAMINING ALLIANCE TREATY RENEGOTIATION

This study investigates the renegotiation of security alliances, specifically the structural conditions surrounding their revision. Although the field of international relations offers a rich discussion of the formation and violation of alliance treaties, few scholars have addressed the reasons why alliance members amend security obligations. After the formation of an alliance, a member may become dissatisfied owing to changes in the external and domestic security environments. A failure to address this discontent increases the risk of alliance breakdown. Members manage their alliance relationship through a negotiation process or intra-alliance bargaining in the search for a new arrangement that can endure. Factors that help to show commitment to the alliance and communicate a set of feasible solutions are crucial if members are to find a mutually acceptable arrangement. By taking these factors into account, allies are more likely to revise an existing treaty. Examining a set of bilateral alliances dating from 1945 to 2001, this research demonstrates that public requests for renegotiation compel allies to change the status quo. It is found that alliance-related fixed assets and the formation of external alliances increase the likelihood of treaty revision, though institutionalization of an alliance does not help to resolve interest divergence. In addition, this study examines the strategy of delay in intra-alliance bargaining. Allies may postpone a dispute by ignoring it while working to maintain the alliance. Tension among allies thus increases, but the alliance endures. I examine three alliances in order to illustrate this renegotiation process. Among these, the Anglo-Japanese alliance demonstrates two successful renegotiations that prolonged a wavering alliance relationship; the Sino-Soviet alliance is an example of failure owing to the lack of substantive cooperation; and the US-Taiwan alliance during the 1970s demonstrates successful use of a strategy of delay that appeases a dissatisfied member.

States' Responding Behavior in Conflict: Asymmetric Response and Strategic Conflict Avoidance

State responses to external threats and aggression are studied with focus on two different rationales: (1) to make credible deterrent threats to avoid being exploited, and (2) to minimize the risk of escalation to unwanted war. Given external aggression, the target state's responding behavior has three possibilities: concession (under-response), reciprocation, and escalation. This study focuses on the first two possibilities and investigates how the strategic nature of crisis interaction can explain the intentional choice of concession or avoidance of retaliation. I build a two-level bargaining model that accounts for the domestic bargaining situation between the leader and the challenger for each state. The model's equilibrium shows that the responding behavior is determined not only by inter-state level variables (e.g. balance of power between two states, or cost of war that each state is supposed to pay), but also the domestic variables of both states. Next, the strategic interaction is rationally explained by the model: as the responding state believes that the initiating state has strong domestic challenges and, hence, the aggression is believed to be initiated for domestic political purposes (a rally-around-the-flag effect), the response tends to decrease. The concession is also predicted if the target state leader has strong bargaining power against her domestic challengers \emph{and} she believes that the initiating leader suffers from weak domestic standing. To test the model's prediction, I conduct a lab experiment and case studies. The experimental result shows that under an incentivized bargaining situation, individual actors are observed to react to hostile action as the model predicts: if the opponent is believed to suffer from internally driven difficulties, the subject will not punish hostile behavior of the other player as severely as she would without such a belief. The experiment also provides supporting evidence for the choice of concession: when the player finds herself in a favorable situation while the other has disadvantages, the player is more likely to make concessions in the controlled dictator game. Two cases are examined to discuss how the model can explain the choice of either reciprocation or concession. From personal interviews and fieldwork in South Korea, I find that South Korea's reciprocating behavior during the 2010 Yeonpyeong Island incident is explained by a combination of `low domestic power of initiating leader (Kim Jong-il)' and `low domestic power of responding leader (Lee Myung-bak).' On the other hand, the case of EC-121 is understood as a non-response or concession outcome. Declassified documents show that Nixon and his key advisors interpreted the attack as a result of North Korea's domestic political instabilities (low domestic power of initiating leader) and that Nixon did not have difficulties at domestic politics during the first few months of his presidency (high domestic power of responding leader).