Introduction: The Next Wave of Enlargement: The European Union and Southeast Europe after 2004

First paragraph: The completion of the Central and Eastern European accession process into the European Union (EU) in 2004 can in broad terms be taken to have been a considerable success in generating an enlarged European zone of peace and stability. But the experience of post-communist transformation within this region is in stark contrast to that in South East Europe where the collapse of the Yugoslav Federation at the end of the Cold War unleashed a bloody and devastating conflict which necessitated the military engagement of the international community and culminated in a NATO-led military intervention into Kosovo in 1999. Although the EU has aspirations to develop a military dimension to its external identity, its international presence continues to be articulated predominantly through soft power e.g. diplomatic, economic and normative foreign policy instruments. The next wave of EU enlargement – if and when it happens - therefore represents a crucial contribution both to the continued creation of a stable European Order and the credibility of the EU as an effective international actor

Scalable, biofunctional, ultra-stable nano- bio- composite materials containing living cells

Three-dimensional encapsulation of cells within nanostructured silica gels or matrices enables applications as diverse as biosensors, microbial fuel cells, artificial organs, and vaccines. It also allows study of individual cell behaviors. Recent progress has improved the performance and flexibility of cellular encapsulation, yet there remains a need for robust scalable processes for large format production of cell-encapsulating materials. Here, we detail two novel techniques, that enable the large-scale production of functional Nano-Bio-Composites (NBCs) containing living cells within ordered 3-D lipid/silica nanostructures: 1) thick-casting and 2) spray drying. Furthermore, we detail a third technique for material scaling in which aqueous, silicate-based gel monoliths encapsulate biofunctional yeast or bacteria. Both dry processes are demonstrated to work with multiple cell types and result in dry powders exhibiting a unique combination of properties including: highly ordered 3-D nanostructure, extended lipid fluidity, tunable macro-morphologies and aerodynamic diameters, and unexpectedly high physical strength. Nanoindentation of the encasing nanostructure revealed Young’s modulus and hardness of 13 and 1.4 GPa respectively, which was unexpected considering the low processing conditions. We hypothesized and confirmed that NBC-encapsulated cells would remain viable for extended periods of time under elevated aging conditions. We attribute this due to the high material strength as observed with nanoindentation, which would prevent cell growth and force bacteria into viable but not culturable (VBNC) states. In concordance with the VBNC state, cellular ATP levels remained elevated even over eight months confirming temperature stable, viable cells. However, their ability to undergo resuscitation and enter growth phase greatly decreased with time in the VBNC state. A quantitative method of determining resuscitation frequencies was developed and showed that, after 36 weeks in an NBC-induced VBNC state, less than 1 in 10,000 cells underwent resuscitation. We verify the VBNC phenotype in gel-encapsulated cells by studying cellular RNA expression levels. These latent behaviors are further demonstrated with an in-vivo immunological study in which mice, immunized with NBCs containing the vaccine Bacillus Calmette-Guérin, were observed to be immunized against a latent form of Tuberculosis. This finding is, in our understanding, the first demonstration of a latent disease-specific live cell immunotherapy. The NBC platform production of industrially scalable quantities of VBNC cells is of interest for research in bacterial persistence and screening of drugs targeting such cells. NBC’s may also enable long-term preservation of living cells for applications in cell-based sensing and the packaging and delivery of live-cell vaccines. Moreover, our methodology represents a novel process for preparing formulations of latent cells in-silico, which could find application in basic cellular research and for the development of a latent-specific vaccine

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis

Pharmaceutical screen identifies novel target processes for activation of autophagy with a broad translational potential

Autophagy is a conserved homeostatic process active in all human cells and affecting a spectrum of diseases. Here we use a pharmaceutical screen to discover new mechanisms for activation of autophagy. We identify a subset of pharmaceuticals inducing autophagic flux with effects in diverse cellular systems modelling specific stages of several human diseases such as HIV transmission and hyperphosphorylated tau accumulation in Alzheimer’s disease. One drug, flubendazole, is a potent inducer of autophagy initiation and flux by affecting acetylated and dynamic microtubules in a reciprocal way. Disruption of dynamic microtubules by flubendazole results in mTOR deactivation and dissociation from lysosomes leading to TFEB (transcription factor EB) nuclear translocation and activation of autophagy. By inducing microtubule acetylation, flubendazole activates JNK1 leading to Bcl-2 phosphorylation, causing release of Beclin1 from Bcl-2-Beclin1 complexes for autophagy induction, thus uncovering a new approach to inducing autophagic flux that may be applicable in disease treatment

What is the cost of a healthy diet? Using diet data from the UK Women's Cohort Study

Background A healthy diet is important to promote health and well-being while preventing chronic disease. However, the monetary cost of consuming such a diet can be a perceived barrier. This study will investigate the cost of consuming a range of dietary patterns.Methods A cross-sectional analysis, where cost of diet was assigned to dietary intakes recorded using a Food Frequency Questionnaire. A mean daily diet cost was calculated for seven data-driven dietary patterns. These dietary patterns were given a healthiness score according to how well they comply with the UK Department of Health's Eatwell Plate guidelines. This study involved �+35 000 women recruited in the 1990s into the UK Women's Cohort Study.Results A significant positive association was observed between diet cost and healthiness of the diet (p for trend >0.001). The healthiest dietary pattern was double the price of the least healthy, -�6.63/day and -�3.29/day, respectively. Dietary diversity, described by the patterns, was also shown to be associated with increased cost. Those with higher education and a professional or managerial occupation were more likely to consume a healthier diet.Conclusions A healthy diet is more expensive to the consumer than a less healthy one. In order to promote health through diet and reduce potential inequalities in health, it seems sensible that healthier food choices should be made more accessible to al

13[C]-Urea Breath Test as a Novel Point-of-Care Biomarker for Tuberculosis Treatment and Diagnosis

BACKGROUND: Pathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers. METHODOLOGY/PRINCIPAL FINDINGS: We tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [(13)C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of delta(13)CO(2) formation were determined. Samples obtained prior to inoculation served as control samples for background (13)CO(2) conversion in the rabbit model. (13)CO(2), from metabolic conversion of [(13)C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of (13)CO(2) formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of (13)CO(2) formation. CONCLUSIONS/SIGNIFICANCE: Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the delta(13)CO(2) signal from urease-positive gastrointestinal organisms

Progress in melanoma modeling in vitro

Melanoma is one of the most studied neoplasia, although laboratory techniques used for investigating this tumor are not fully reliable. Animal models may not predict the human response due to differences in skin physiology and immunity. In addition, international guidelines recommend to develop processes that contribute to the reduction, refinement and replacement of animals for experiments (3Rs). Adherent cell culture has been widely used for the study of melanoma to obtain important information regarding melanoma biology. Nonetheless, these cells grow in adhesion on the culture substrate which differs considerably from the situation in vivo. Melanoma grows in a 3D spatial conformation where cells are subjected to a heterogeneous exposure to oxygen and nutrient. In addition, cell-cell and cell-matrix interaction play a crucial role in the pathobiology of the tumor as well as in the response to therapeutic agents. To better study melanoma new techniques, including spherical models, tumorospheres, and melanoma skin equivalents have been developed. These 3D models allow to study tumors in a microenvironment that is more close to the in vivo situation, and are less expensive and time consuming than animal studies. This review will also describe the new technologies applied to skin reconstructs such as organ-on-a-chip that allows skin perfusion through microfluidic platforms. 3D in vitro models, based on the new technologies, are becoming more sophisticated, representing at a great extent the in vivo situation, the "perfect" model that will allow less involvement of animals up to their complete replacement, is still far from being achieved. This article is protected by copyright. All rights reserved