Real-time intermembrane force measurements and imaging of lipid domain morphology during hemifusion

Membrane fusion is the core process in membrane trafficking and is essential for cellular transport of proteins and other biomacromolecules. During protein-mediated membrane fusion, membrane proteins are often excluded from the membrane-membrane contact, indicating that local structural transformations in lipid domains play a major role. However, the rearrangements of lipid domains during fusion have not been thoroughly examined. Here using a newly developed Fluorescence Surface Forces Apparatus (FL-SFA), migration of liquid-disordered clusters and depletion of liquid-ordered domains at the membrane-membrane contact are imaged in real time during hemifusion of model lipid membranes, together with simultaneous force-distance and lipid membrane thickness measurements. The load and contact time-dependent hemifusion results show that the domain rearrangements decrease the energy barrier to fusion, illustrating the significance of dynamic domain transformations in membrane fusion processes. Importantly, the FL-SFA can unambiguously correlate interaction forces and in situ imaging in many dynamic interfacial systems.open0

Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism

Cholesterol Induces Specific Spatial and Orientational Order in Cholesterol/Phospholipid Membranes

In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterol's specific ordering and packing capability have remained unresolved

Cholesterol Induces Specific Spatial and Orientational Order in Cholesterol/Phospholipid Membranes

In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterol's specific ordering and packing capability have remained unresolved

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Impact of structural habitat modifications in coastal temperate systems on fish recruitment: A systematic review

none6Background: Shallow nearshore marine ecosystems are changing at an increasing rate due to a range of human activities such as urbanisation and commercial development. As a result, an increasing number of structural modifications occur in coastal nursery and spawning habitats of fish. Concomitant to this increase, there have been declines in many coastal fish populations and changes in the composition of fish communities. As requested by Swedish stakeholders, this review aimed to synthesise scientific evidence of the impact on fish recruitment of structural modifications in temperate coastal areas. Methods: We searched for peer-reviewed and grey literature on such impacts in English, Dutch, Danish, Finnish, German, Swedish and Spanish. Searches were performed in bibliographic databases, specialist websites, bibliographies of review articles. We also contacted stakeholder to find relevant literature. Eligible studies included small- and large-scale field studies in marine systems and large lakes (> 10,000 km(2)) in temperate regions of the Northern and Southern Hemispheres. Included replicated comparisons of fish recruitment between altered and unaltered control areas, comparisons before and after an alteration, or both. Relevant outcomes (response variables) included measures of recruitment defined as abundance of juvenile fish in coastal habitats. All fish species were considered. Articles were screened for eligibility by title, abstract and full text. Eligible studies were critically appraised based on their external and internal validity. From each eligible study of sufficient validity, we extracted information on study design, measured outcomes, exposure, type of comparator, effect modifiers and study findings. Study findings were synthesised narratively. Results: We searched for eligible studies in 15 databases, 24 specialist websites, Google Scholar, and bibliographies of 11 review articles. The review finally included 37 studies that were eligible and of sufficient validity to be considered for final synthesis. Most studies (23 of 37) were from the Northern Hemisphere. Studies varied in design, spatial resolution, target fish species, and type of structural habitat change. This high level of variation did not allow for a quantitative synthesis and prevented us from drawing general conclusions on the impact of structures or structural modifications on fish recruitment. In this review we provide a narrative synthesis of the evidence base and classify eligible studies into six categories (based on type of exposure and comparator). The categories are as follows: the impacts on fish recruitment of: (1) artificial structures in coastal areas, (2) structures designed as fish attractors, (3) large scale urban sprawl, (4) 'novel' habitats, (5) habitat loss, and (6) restoration. Conclusions: This review revealed a very limited evidence base for how structural modifications and marine urban sprawl can affect fish recruitment. Thus, there is a substantial mismatch between stakeholder needs and research evidence. Further, the impact and ecological performance of artificial structures depend both on context and species. Clearly, there is a need for more research on the subject, especially on long-term consequences at larger spatial scales.mixedMacura B.; Bystrom P.; Airoldi L.; Eriksson B. K.; Rudstam L.; Stottrup J. G.Macura, B.; Bystrom, P.; Airoldi, L.; Eriksson, B. K.; Rudstam, L.; Stottrup, J. G

Upregulation of Receptor Tyrosine Kinase Activity and Stemness as Resistance Mechanisms to Akt Inhibitors in Breast Cancer

The PI3K/Akt pathway is frequently deregulated in human cancers, and multiple Akt inhibitors are currently under clinical evaluation. Based on the experience from other molecular targeted therapies, however, it is likely that acquired resistance will be developed in patients treated with Akt inhibitors. We established breast cancer models of acquired resistance by prolonged treatment of cells with allosteric or ATP-competitive Akt inhibitors. Phospho-Receptor tyrosine kinase (Phospho-RTK) arrays revealed hyper-phosphorylation of multiple RTKS, including EGFR, Her2, HFGR, EhpB3 and ROR1, in Akt-inhibitor-resistant cells. Importantly, resistance can be overcome by treatment with an EGFR inhibitor. We further showed that cancer stem cells (CSCs) are enriched in breast tumor cells that have developed resistance to Akt inhibitors. Several candidates of CSC regulators, such as ID4, are identified by RNA sequencing. Cosmic analysis indicated that sensitivity of tumor cells to Akt inhibitors can be predicted by ID4 and stem cell/epithelial–mesenchymal transition pathway targets. These findings indicate the potential of targeting the EGFR pathway and CSC program to circumvent Akt inhibitor resistance in breast cancer