Study of P. falciparum-infected erythrocytes and induced anisotropies under optical and fluid forces

Background & objectives: The effect of P. falciparum on erythrocytes has been studied for a long time at the population level but actual studies at the single cell level remain largely unexplored. The aim of this study was to address the host-parasite relationship at the single cell level under two different kinds of forces, an optical force and a fluid force. The questions addressed were about the basic host-parasite interactions, but our findings have larger implications in diverse fields of parasite biology. Methods: Erythrocytes were monitored under optical forces (using optical tweezers) and fluid forces (using microfluidic chambers) and dynamical images were captured in real-time video clips. These videos were then split into their respective frames so as to yield temporal information and various parameters pertaining to membrane structure, ionic imbalance and interaction with different forces were studied. Results: The results of this study mainly bring to fore the inherent differences between infected and normal cell populations at the single cell level under various external forces. We probed three different criteria folding times, rotation speeds and rolling frequency to show inherent difference in various cell populations and also the dependence of the above to the cycle of the parasite. Interpretation & conclusion: This study portrays the importance of single cell observations pertaining to the host-parasite relationship. It shows the effect the malarial parasite has on erythrocytes and how the intrinsic property of the infected and its neighbouring uninfected cells change as compared to normal erythrocytes. There are thus implications in the fields of cytoadherence, parasite invasions and host immune evasion

Plasmodium-infected red blood cells exhibit enhanced rolling independent of host cells and alter flow of uninfected red cells

The pathogenicity of Plasmodium falciparum results from its unique ability to adhere to endothelium and uninfected erythrocytes. It is, therefore, important to understand the events leading to flowing blood cells undergoing such adhesion. Largely based on the leukocyte adhesion model, it is postulated that the slowing down (rolling) of Plasmodium-infected red blood cells (PRBCs) is initiated by interactions between certain host adhesion molecules and the parasite proteins. In this article we present data demonstrating that PRBCs do not require the presence of host adhesion molecules to slow down and roll. In a synchronized culture, the proportion of slow-flowing cells increased with parasite development and was highest at the trophozoite stage. We also observed that the uninfected red cells (URBCs), originating from a parasite culture containing PRBCs, were also inherently slower compared to malaria-unexposed normal red blood cells (NRBCs). NRBCs became slower upon incubation with supernatant taken from a parasite culture. However, such an effect was transient and the NRBCs reverted to their normal flow speed within 12 h upon withdrawal of culture supernatant. Based on our observations, we suggest that the higher propensity of PRBCs and URBCs to slow down is due to inherent structural anisotropy and altered membrane rigidity. Thus the initial events leading to the slowing down of malaria-infected blood cells appear to be different from those occurring during leukocyte adhesion

Human Rad52 binding renders ssDNA unfolded: image and contour length analyses by Atomic Force Microscopy

Atomic force microscopy imaging has been used to study the changes associated with human Rad52 (HsRad52) protein in solution, in dried state as well as following ssDNA (linear and circular) binding. In the dried state, the free protein exists predominantly as a characteristic panoply of novel trifoliate forms. However, in solution, the level of trifoliates diminishes significantly. Height analyses of either form reveal two categories: smaller (~ 3-5 nm) and larger ((~ 10-12 nm) particles, perhaps related to sub-heptameric and heptameric forms respectively. Interestingly, binding of the protein to linear ssDNA smoothly extends and unfolds the naked DNA. Contour length measurements performed on several individual circular ssDNA/nucleoprotein complexes reveal marked (about threefold) extension of naked ssDNA, following HsRad52 binding. We speculate that the alignment of HsRad52 on ssDNA into a smoothly extended and unfolded strand from that of highly compact morphology of naked ssDNA, may have bearing on the recombination function of HsRad52 protein

Assembling Neurospheres: Dynamics of Neural Progenitor/Stem Cell Aggregation Probed Using an Optical Trap

Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (∼5 s) and most probable minimum distance of approach (4–6 µm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (∼18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and “stemness”. Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion

Torque-generating malaria-infected red blood cells in an optical trap

We have used optical tweezers to trap normal and Plasmodiuminfected red blood cells (iRBCs). Two different facets of the behavior of RBCs in infrared light fields emerge from our experiments. Firstly, while the optical field modifies both types of RBCs in the same fashion, by folding the original biconcave disk into a rod-like shape, iRBCs rotate with linearly polarized light whereas normal RBCs do not. Secondly, and in the context of known molecular motors, our measurements indicate that the torque of rotating iRBCs is up to three orders of magnitude larger

Suppression of ultrafast supercontinuum generation in a salivary protein

The first studies of the propagation of ultrafast (<45 fs) pulses of intense infrared light through protein media reveal that supercontinuum (white light) generation is severely suppressed in the presence of the protein α -amylase, a potential stress marker in human saliva. The continuum suppression capacity is attributed to the electron scavenging property of the protein

Strategic Performance Measurement Using Balanced Scorecard: A Case of Machine Tool Industry

This paper focuses on implementation, monitoring, and application of balanced scorecard (BSC) techniques in an organization involved in providing machine tool solutions to the industrial sector. The growth of the company considered in real time constituted improvements of both top and bottom lines. In the industry under consideration, it was observed that in our company, the top line was steadily growing but not the bottom line. This is when we started getting down to brass tacks and strategically focusing on growth in overall profits of the company. This included growing revenues by improving of EBITDA (earnings before interests, taxes, depreciation, and amortization) and by increasing efficiency (i.e., cutting costs). These improvements were implemented by chalking out a comprehensive BSC designed to suit the machine tool industry. The four perspectives of the management, namely, internal business process, organizational learning, financial perspective, and customer perspective, have been considered lucidly and enunciate the parameters that affect the BSC very aptly. The BSC designed considered 9 objectives and 27 relative measures of these factors to quantify the various quantitative and qualitative dimensions that affect the company’s performance. A Balanced Lean Index (BL Score) was used to measure the results for company X