Cell migration through 3D confining pores: speed accelerations by deformation and recoil of the nucleus

Directional cell migration in dense three-dimensional (3D) environments critically depends upon shape adaptation and is impeded depending on the size and rigidity of the nucleus. Accordingly, the nucleus is primarily understood as a physical obstacle, however, its pro-migratory functions by step-wise deformation and reshaping remain unclear. Using atomic force spectroscopy, timelapse fluorescence microscopy and shape change analysis tools, we determined nuclear size, deformability, morphology and shape change of HT1080 fibrosarcoma cells expressing the Fucci cell cycle indicator or being pre-treated with chromatin-decondensating agent TSA. We show oscillating peak accelerations during migration through 3D collagen matrices and microdevices that occur during shape reversion of deformed nuclei (recoil), and increase with confinement. During G1 cell cycle phase, nucleus stiffness was increased and yielded further increased speed fluctuations together with sustained cell migration rates in confinement as compared to interphase populations, or to periods of intrinsic nuclear softening in the S/G2 cell cycle phase. Likewise, nuclear softening by pharmacological chromatin decondensation or after lamin A/C depletion reduced peak oscillations in confinement. In conclusion, deformation and recoil of the stiff nucleus contributes to saltatory locomotion in dense tissues

‘The war is a money making show’: Working-Class Attitudes to World War II and Australian Nationalism

This paper will address the conference themes of ‘class, power and social structure’ through examining industrial and ideological conflict during World War II. The paper will also address the theme of ‘class and culture’ through an examination of working-class cultural expression as a means of resistance to the government’s wartime offensive. What is overlooked in most histories of World War II is the working-class experience of the war and their understanding of nationalism, particularly as nationalism was cynically exploited by the government to undermine working-class identity and solidarity. The paper will investigate the experience of one of the most militant sections of the Australian working class: the Miners. Primary source material such as the Miners’ journal Common Cause and union records reveal opposition to the war and a much more ambiguous attitude to the national sentiment used to justify Australia’s involvement. The Miners provide an interesting case study as the union was led by the Communist Party. Therefore the union leadership initially opposed to the war then became enthusiastic supporters when Russia entered the war on the allied side. It is clear that the Miners’ union leadership found it difficult to convince the rank and file to support the war. The paper will focus upon rank and file attitudes to the war and Australian nationalism particularly during times of industrial unrest.The symposium is organised on behalf of AAHANZBS by the Business and Labour History Group, The University of Sydney, with the financial support of the University’s Faculty of Economics and Business

A systematic review and meta-analysis of F-18-fluoro-D-deoxyglucose positron emission tomography interpretation methods in vascular graft and endograft infection

OBJECTIVE: Vascular graft and endograft infection (VGEI) has high morbidity and mortality rates. Diagnosis is complicated since symptoms vary and can be non-specific. A recent meta-analysis identified the use of 18F-fluoro-D-deoxyglucose positron emission tomography-computed tomography (18F-FDG PET(/CT)) as the most valuable tool for diagnosing VGEI and favorable to computed tomography as the current standard. However, the availability and varied use of several interpretation methods, without consensus on which interpretation method is best, complicates clinical use. The aim of this study was to evaluate the diagnostic performance of different interpretation methods of 18F-FDG PET(/CT) in diagnosing VGEI. METHODS: A systematic review was performed according to the PRISMA guidelines. Data sources included PubMed/Medline, Embase, and Cochrane. A meta-analysis was conducted on the different interpretation methods for 18F-FDG PET(/CT) in diagnosing VGEI, including visual FDG uptake intensity, visual FDG uptake pattern, and quantitative SUVmax. RESULTS: Out of 613 articles, 13 were included-10 prospective and 3 retrospective articles. The FDG uptake pattern method (I2 26.2%) showed negligible heterogeneity, while the FDG uptake intensity (I2 42.2%) and SUVmax (I2 42.1%) methods both showed moderate heterogeneity. The pooled sensitivity for FDG uptake intensity was 0.90 (95% CI: 0.79-0.96), for uptake pattern 0.94 (95% CI: 0.89-0.97), and for the SUVmax method 0.95 (95% CI: 0.76-0.99). The pooled specificity for FDG uptake intensity was 0.59 (95% CI: 0.38-0.78), whereas for FDG uptake pattern it was 0.81 (95% CI: 0.71-0.88) and for SUVmax it was 0.77 (95% CI: 0.63-0.87). The uptake pattern interpretation method demonstrated the best positive and negative post-test probability-82% and 10%, respectively. CONCLUSION: This meta-analysis identified the FDG uptake pattern as the most accurate assessment method of 18F-FDG PET(/CT) for diagnosing VGEI. The optimal SUVmax cutoff, depending on the vendor, demonstrated strong sensitivity and moderate specificity

Biophysical Characterization of CD6—TCR/CD3 Interplay in T Cells

Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts. In this study, we report the use of different experimental set-ups analyzed with microscopy techniques to study the dynamics and stability of CD6-TCR/CD3 interaction dynamics and stability during IS formation in more detail. We exploited antibody spots, created with microcontact printing, and antibody-coated beads, and could demonstrate that CD6 and the TCR/CD3 complex co-localize and are recruited into a stimulatory cluster on the cell surface of T cells. Furthermore, we demonstrate, for the first time, that CD6 forms microclusters co-localizing with TCR/CD3 microclusters during IS formation on supported lipid bilayers. These co-localizing CD6 and TCR/CD3 microclusters are both radially transported toward the center of the IS formed in T cells, in an actin polymerization-dependent manner. Overall, our findings further substantiate the role of CD6 during IS formation and provide novel insight into the dynamic properties of this CD6-TCR/CD3 complex interplay. From a methodological point of view, the biophysical approaches used to characterize these receptors are complementary and amenable for investigation of the dynamic interactions of other membrane receptors

Locomotion disorders and skin and claw lesions in gestating sows housed in dynamic versus static groups

Lameness and lesions to the skin and claws of sows in group housing are commonly occurring indicators of reduced welfare. Typically, these problems are more common in group housing than in individual housing systems. Group management type (dynamic versus static) and stage of gestation influence the behavior of the animals, which in turn influences the occurrence of these problems. The present study compared prevalence, incidence and mean scores of lameness and skin and claw lesions in static versus dynamic group housed sows at different stages of gestation during three consecutive reproductive cycles. A total of 10 Belgian sow herds were monitored; 5 in which dynamic groups and 5 in which static groups were utilized. All sows were visually assessed for lameness and skin lesions three times per cycle and the claws of the hind limbs were assessed once per cycle. Lameness and claw lesions were assessed using visual analogue scales. Static groups, in comparison with dynamic groups, demonstrated lower lameness scores (P<0.05) and decreased skin lesion prevalence (24.9 vs. 47.3%, P<0.05) at the end of gestation. There was no difference between treatment group regarding claw lesion prevalence with 75.5% of sows demonstrating claw lesions regardless of group management. Prevalences of lameness (22.4 vs. 8.9%, P<0.05) and skin lesions (46.6 vs. 4.4%, P<0.05) were highest during the group-housed phase compared to the individually housed phases. Although the prevalence of lameness and skin lesions did not differ three days after grouping versus at the end of the group-housing phase, their incidence peaked during the first three days after moving from the insemination stalls to the group. In conclusion, the first three days after grouping was the most risky period for lameness incidence, but there was no significant difference between static or dynamic group management

Impaired Vascular Contractility and Aortic Wall Degeneration in Fibulin-4 Deficient Mice: Effect of Angiotensin II Type 1 (AT1) Receptor Blockade

Medial degeneration is a key feature of aneurysm disease and aortic dissection. In a murine aneurysm model we investigated the structural and functional characteristics of aortic wall degeneration in adult fibulin-4 deficient mice and the potential therapeutic role of the angiotensin (Ang) II type 1 (AT1) receptor antagonist losartan in preventing aortic media degeneration. Adult mice with 2-fold (heterozygous Fibulin-4+/R) and 4-fold (homozygous Fibulin-4R/R) reduced expression of fibulin-4 displayed the histological features of cystic media degeneration as found in patients with aneurysm or dissection, including elastin fiber fragmentation, loss of smooth muscle cells, and deposition of ground substance in the extracellular matrix of the aortic media. The aortic contractile capacity, determined by isometric force measurements, was diminished, and was associated with dysregulation of contractile genes as shown by aortic transcriptome analysis. These structural and functional alterations were accompanied by upregulation of TGF-β signaling in aortas from fibulin-4 deficient mice, as identified by genome-scaled network analysis as well as by immunohistochemical staining for phosphorylated Smad2, an intracellular mediator of TGF-β. Tissue levels of Ang II, a regulator of TGF-β signaling, were increased. Prenatal treatment with the AT1 receptor antagonist losartan, which blunts TGF-β signaling, prevented elastic fiber fragmentation in the aortic media of newborn Fibulin-4R/R mice. Postnatal losartan treatment reduced haemodynamic stress and improved lifespan of homozygous knockdown fibulin-4 animals, but did not affect aortic vessel wall structure. In conclusion, the AT1 receptor blocker losartan can prevent aortic media degeneration in a non-Marfan syndrome aneurysm mouse model. In established aortic aneurysms, losartan does not affect aortic architecture, but does improve survival. These findings may extend the potential therapeutic application of inhibitors of the renin-angiotensin system to the preventive treatment of aneurysm disease

A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4 + T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention

Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E2 (PGE2). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE2 causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE2-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses

Force spectroscopy to understand cell surface receptor interactions in the immune system.

Contains fulltext : 77577.pdf (publisher's version ) (Open Access)Since the invention of the atomic force microscope (AFM) in 1986, the application of the instrument has shifted from a pure physicist’s tool to a useful instrument for physicists, chemists and biologists. The AFM is not a classical microscope in the sense that light or electrons are used to obtain an image, it is rather a mechanical microscope. The imaging is purely based on a physical probing of the surface topography by raster scanning (zigzag movements) of a sharp tip on a cantilever across the sample. Furthermore, the AFM can probe forces with ultra-high sensitivity by moving the cantilever in the vertical direction, which is especially interesting for biological applications. The probing of forces with AFM is known as AFM force spectroscopy. These AFM measurements can be performed under physiological conditions and make it possible to study dynamical processes, such as the adhesion of a living cell to an adjacent cell or the extracellular matrix, from the single cell to single molecular level. In the immune system diverse dynamical processes, such as the recognition of a pathogen by a dendritic cell (DC) or the transmigration of a leukocyte through the endothelium of a blood vessel, happen all the time throughout the human body. In the latter case during initial binding of these cells, bonds between different cell adhesion molecules (CAMs) are formed. These interactions have to be highly dynamic to withstand forces induced by for example the blood flow. These CAM bonds associate and dissociate at rates that change considerably under conditions of cell stress. The combination of molecular cell biology and AFM single molecule force spectroscopy provides a powerful tool to explore the complexity of these cell adhesion processes. In this thesis, we explored the versatility of the AFM in the context of biomaterial science, nano-chemistry and cell biology. The high spatial resolution of the AFM was exploited to measure and manipulate samples at the nanoscale. Moreover, the high force sensitivity of the AFM was applied to measure adhesive properties of CAMs on cells and to address the two distinct adhesive regulation processes of a CAM: affinity and avidity.RU Radboud Universiteit Nijmegen, 21 juni 2010Promotores : Speller, S.E., Figdor, C.G. Co-promotor : Cambi, A.176 p