Force-induced unfolding of fibronectin in the extracellular matrix of living cells.

Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes.

Micro-well arrays for 3D shape control and high resolution analysis of single cells.

In addition to rigidity, matrix composition, and cell shape, dimensionality is now considered an important property of the cell microenvironment which directs cell behavior. However, available tools for cell culture in two-dimensional (2D) versus three-dimensional (3D) environments are difficult to compare, and no tools exist which provide 3D shape control of single cells. We developed polydimethylsiloxane (PDMS) substrates for the culture of single cells in 3D arrays which are compatible with high-resolution microscopy. Cell adhesion was limited to within microwells by passivation of the flat upper surface through 'wet-printing' of a non-fouling polymer and backfilling of the wells with specific adhesive proteins or lipid bilayers. Endothelial cells constrained within microwells were viable, and intracellular features could be imaged with high resolution objectives. Finally, phalloidin staining of actin stress fibers showed that the cytoskeleton of cells in microwells was 3D and not limited to the cell-substrate interface. Thus, microwells can be used to produce microenvironments for large numbers of single cells with 3D shape control and can be added to a repertoire of tools which are ever more sought after for both fundamental biological studies as well as high throughput cell screening assays.

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. I. POLLEN TUBE AND ARCHEGONIAL DEVELOPMENT.

Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) ovules were used to study the method of pollen tube formation and penetration of the nucellus, the movement of the body cell down the pollen tube and development of the archegonia. No pollination drop forms but nucellar tip cells produce a minute secretion that may initiate pollen tube formation. Pollen tubes penetrate the nucellus causing degeneration of nucellar cells in contact with the pollen tube tip. The body cell becomes highly lobed and the tube cytoplasm forms thin sheets between the lobes. This may be the mechanism by which the large body cell is pulled down the narrow pollen tube. Body cell plastids and mitochondria remain unaltered during pollen tube growth, whereas tube cell organelles show signs of degeneration. The pollen tube penetrates the megaspore wall and settles in the archegonial chamber. During pollen elongation and pollen tube growth the egg matured. Egg cell plastids were transformed into large inclusions which filled the periphery of the egg while mitochondria migrated to the perinuclear zone. The neck cells, ventral canal cell and archegonial jacket cells are described. The significance of the body cell and egg cell ultrastructure is discussed in light of recent restriction fragment length polymorphism studies of plastid and mitochondrial inheritance in the Pinaceae.

Travellers as sentinels: Assaying the worldwide distribution of polymorphisms associated with artemisinin combination therapy resistance in Plasmodium falciparum using malaria cases imported into Scotland.

There is growing evidence that Plasmodium falciparum parasites in southeastern Asia have developed resistance to artemisinin combination therapy. The resistance phenotype has recently been shown to be associated with four single nucleotide polymorphisms in the parasite's genome. We assessed the prevalence of two of these single nucleotide polymorphisms in P. falciparum parasites imported into Scotland between 2009 and 2012, and in additional field samples from six countries in southeastern Asia. We analysed 28 samples from 11 African countries, and 25 samples from nine countries in Asia/southeastern Asia/Oceania. Single nucleotide polymorphisms associated with artemisinin combination therapy resistance were not observed outside Thailand and Cambodia.

Imported malaria in Scotland--an overview of surveillance, reporting and trends.

BACKGROUND: Imported malaria cases continue to occur and are often underreported. This study assessed reporting of malaria cases and their characteristics in Scotland. METHODS: Cases were identified at the study sites of Aberdeen, Edinburgh, Glasgow and Inverness. The number of cases identified in the period 2003-2008 was compared to surveillance databases from Health Protection Scotland (HPS) and the Malaria Reference Laboratory (MRL). Case characteristics were recorded and analysed. RESULTS: Of 252 cases of malaria diagnosed and treated, an estimated 235 (93.3%) were reported to the MRL. Between 2006 and 2008, 114 of 126 cases (90.5%) were reported to HPS. Plasmodium falciparum caused 173 cases (68.7%). Business and professional travel accounted for 35.3% of cases (higher in Aberdeen), followed by visiting friends and relatives (33.1%) and holiday makers (25.5%). The majority of infections were imported from West Africa and 65.7% of patients for whom data on prophylaxis was available had taken no or inappropriate prophylaxis. CONCLUSIONS: Reporting of malaria in Scotland can be improved. There is a continued need to optimise preventive measures and adherence to chemoprophylaxis amongst business travellers, those visiting friends and relatives, and holiday makers in endemic countries in order to reduce imported malaria cases.

HIV-1-infected CD8+CD4+ T cells decay in vivo at a similar rate to infected CD4 T cells during HAART.

OBJECTIVES: To investigate the potential for CD4+CD8+ T cells [CD8 double positive (CD8 DP)] T cells to form a reservoir of HIV-1 following HAART through measurement of the rate of decay of infected CD4/CD8 DP T cells. METHODS: HIV-1 proviral loads in highly pure CD4 and CD8 DP T cells were determined for study subjects before and after 200-400 days of therapy and HIV-1 DNA decay rates were calculated. RESULTS: Before therapy, HIV-1 proviral load in CD8 DP correlated negatively with CD4 cell count. Decay rates of HIV-1-infected CD4 and CD8 DP T cells were similar. Rates for CD8 DP T cells correlated with the time to suppression of viral replication, whereas no such relationship was true for CD4 cell decay rates. A significant reduction in activated cells was observed for both cell types. The action of HAART on HIV-1 replication was similar for both CD4 cells and CD8 DP T cells, although the rate of clearance of infected CD8 DP T cells appeared more critical for a rapid reduction in plasma viral load. Although the size of the CD8 DP T cell reservoir in peripheral blood was smaller relative to that of CD4 cells, HAART did not completely clear HIV-1 infection from this cell subset. CONCLUSION: This study confirmed that CD8 DP T cells are a major reservoir for HIV-1 in vivo and, therefore, represent a potential reservoir for HIV-1 during HAART, in a manner analogous to that of CD4 T cells.