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Parasitization by malaria-inducing Plasmodium falciparum leads to structural, biochemical, and mechanical modifications to the host red blood cells (RBCs). To study these modifications, we investigate two intrinsic indicators: the refractive index and membrane fluctuations in P. falciparum-invaded human RBCs (Pf-RBCs). We report experimental connections between these intrinsic indicators and pathological states. By employing two noninvasive optical techniques, tomographic phase microscopy and diffraction phase microscopy, we extract three-dimensional maps of refractive index and nanoscale cell membrane fluctuations in isolated RBCs. Our systematic experiments cover all intraerythrocytic stages of parasite development under physiological and febrile temperatures. These findings offer potential, and sufficiently general, avenues for identifying, through cell membrane dynamics, pathological states that cause or accompany human diseases.erythrocyte ͉ febrile temperature ͉ malaria ͉ mechanical properties ͉ optical techniques D uring the intraerythrocytic development, the malaria parasite Plasmodium falciparum causes structural, biochemical, and mechanical changes to host red blood cells (RBCs). Major structural changes include the formation of parasitophorus vacuoles that surround the growing parasite in their host RBCs, loss of cell volume, and the appearance of small, nanoscale protrusions or ''knobs,'' on the membrane surface (1). From the biochemical standpoint, a considerable amount of hemoglobin (Hb) is digested by parasites during intraerythrocytic development and converted into insoluble polymerized forms of heme, known as hemozoin (2, 3). Hemozoin appears as brown crystals in the vacuole of parasite in later maturation stages of P. falciparum-invaded human RBCs (Pf-RBCs).Two major mechanical modifications are loss of RBC deformability (4-6) and increased cytoadherence of the invaded RBC membrane to vascular endothelium and other RBCs (7). These changes lead to sequestration of RBCs in microvasculature in the later stages of parasite development, which is linked to vital organ dysfunction in severe malaria. In the earlier stage, where some loss of deformability occurs, Pf-RBCs continue to circulate in the bloodstream.Membrane dynamics of RBCs can be influenced by human disease states. Fluctuations in phospholipid bilayer and attached spectrin network are known to be altered by cytoskeletal defects, stress, and actin-spectrin dissociations arising from metabolic activity linked to adenosine 5Ј-triphosphate (ATP) concentration (8-12). Proteins transported from invading organisms, such as the virulent malaria-inducing parasite P. falciparum, to specific binding sites in the spectrin network are considered to introduce significant alterations to RBC membrane dynamics and mechanical response (13,14). These changes could provide insights into possible mechanistic pathways in the pathogenesis of malaria, because the parasite alters biophysical properties of RBCs during its intraerythrocyte stage that lasts up to 48 ...

The human red blood cell (RBC) membrane, a fluid lipid bilayer tethered to an elastic 2D spectrin network, provides the principal control of the cell's morphology and mechanics. These properties, in turn, influence the ability of RBCs to transport oxygen in circulation. Current mechanical measurements of RBCs rely on external loads. Here we apply a noncontact optical interferometric technique to quantify the thermal fluctuations of RBC membranes with 3 nm accuracy over a broad range of spatial and temporal frequencies. Combining this technique with a new mathematical model describing RBC membrane undulations, we measure the mechanical changes of RBCs as they undergo a transition from the normal discoid shape to the abnormal echinocyte and spherical shapes. These measurements indicate that, coincident with this morphological transition, there is a significant increase in the membrane's shear, area, and bending moduli. This mechanical transition can alter cell circulation and impede oxygen delivery. promises more sensitive probes of their structure at the nanoscale and suggests new insights into the etiology of a number of human diseases (1, 2). In the healthy individual, these cells withstand repeated, large-amplitude mechanical deformations as they circulate through the microvasculature. Certain pathological conditions such as spherocytosis, malaria, and Sickle cell disease cause changes in both the equilibrium shape and mechanics of RBCs, which impact their transport function. Here we communicate measurements of RBC mechanics that rely on unique experimental and theoretical techniques to characterize the mechanics/rheology of normal and pathological RBCs over a range of length and time scales.Lacking a 3D cytoskeleton, RBCs maintain their shape and mechanical integrity through a spectrin-dominated, triangular 2D network attached to the cytosolic side of their plasma membrane. This semiflexible filament network, along with the surface tension of the bilayer, contributes to the elastic moduli of the composite membrane (3). The fluid lipid bilayer is thought to be the principal contributor to its bending or curvature modulus. Little is known about the molecular and structural transformations that take place in the membrane and spectrin network during the cell's morphological transitions from discocyte (DC, normal shape) to echinocyte (EC, spiculated shape) to spherocyte (SC, nearly spherical) (Figs. 1 A-C), which are accompanied by changes in RBC mechanics.A number of techniques have been used to study the rheology of live cells (2). Micropipette aspiration (4), electric field deformation (5), and optical tweezers (2) provide quantitative information about the shear and bending moduli of RBC membranes in static conditions. However, dynamic, frequencydependent knowledge of RBC mechanics is currently very limited with the notable exception of ref. 6. RBC thermal fluctuations ("flickering") have been studied for more than a century (7) to better understand the interaction between the lipid bilayer and the cytoskeleto...

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Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationaryphase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.Bacteria in nature are often exposed to dramatic fluctuations in external pH that threaten viability. Survival, therefore, depends on the presence of adaptive mechanisms that sense an acidifying environment and coordinate an appropriate molecular response (15,17). Salmonella enterica serovar Typhimurium employs several strategies to avoid or repair damage associated with acid stress. Two major low-pH-inducible systems, known as acid tolerance responses (ATR), have been identified. They are classified based on the growth phase at which each becomes induced. Most studies have focused on the log-phase ATR system induced when exponentially growing cells suddenly undergo a rapid transition to low pH (16). Over 50 acid shock proteins (ASPs) are produced during this response (14). The regulatory genes rpoS, encoding an alternative sigma factor, and fur, encoding the major iron regulator, are required for log-phase acid tolerance and control the production of subsets of the ASPs (15,17,19,33).The second ATR system, referred to as the stationary-phase ATR, is induced by exposing stationary-phase cells to low pH (34). It is distinct from the general stress response system that is induced by entry into stationary phase regardless of the culture pH. The general stress response system requir...

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Lipid droplets (LDs) are subcellular organelles with important roles in lipid storage and metabolism and involved in various diseases including cancer, obesity, and diabetes. Conventional methods, however, have limited ability to provide quantitative information on individual LDs and have limited capability for three-dimensional (3-D) imaging of LDs in live cells especially for fast acquisition of 3-D dynamics. Here, we present an optical method based on 3-D quantitative phase imaging to measure the 3-D structural distribution and biochemical parameters (concentration and dry mass) of individual LDs in live cells without using exogenous labelling agents. The biochemical change of LDs under oleic acid treatment was quantitatively investigated, and 4-D tracking of the fast dynamics of LDs revealed the intracellular transport of LDs in live cells.

Two-dimensional (2D) nanomaterials, such as graphene-based materials and transition metal dichalcogenide (TMD) nanosheets, are promising materials for biomedical applications owing to their remarkable cytocompatibility and physicochemical properties. On the basis of their potent antibacterial properties, 2D materials have potential as antibacterial films, wherein the 2D nanosheets are immobilized on the surface and the bacteria may contact with the basal planes of 2D nanosheets dominantly rather than contact with the sharp edges of nanosheets. To address these points, in this study, we prepared an effective antibacterial surface consisting of representative 2D materials, i.e., graphene oxide (GO) and molybdenum disulfide (MoS), formed into nanosheets on a transparent substrate for real device applications. The antimicrobial properties of the GO-MoS nanocomposite surface toward the Gram-negative bacteria Escherichia coli were investigated, and the GO-MoS nanocomposite exhibited enhanced antimicrobial effects with increased glutathione oxidation capacity and partial conductivity. Furthermore, direct imaging of continuous morphological destruction in the individual bacterial cells having contacts with the GO-MoS nanocomposite surface was characterized by holotomographic (HT) microscopy, which could be used to detect the refractive index (RI) distribution of each voxel in bacterial cell and reconstruct the three-dimensional (3D) mapping images of bacteria. In this regard, the decreases in both the volume (67.2%) and the dry mass (78.8%) of bacterial cells that came in contact with the surface for 80 min were quantitatively measured, and releasing of intracellular components mediated by membrane and oxidative stress was observed. Our findings provided new insights into the antibacterial properties of 2D nanocomposite film with label-free tracing of bacterial cell which improve our understanding of antimicrobial activities and opened a window for the 2D nanocomposite as a practical antibacterial film in biomedical applications.