1. Although influenza kills about a half million people each year, even after excluding pandemics, there is only one set of antiviral drugs: neuraminidase inhibitors. By using a new approach utilizing giant unilamellar vesicles and infectious X-31 influenza virus, and testing for the newly identified pore intermediate of membrane fusion, we observed 3087% poration, depending upon lipid composition. Testing the hypothesis that spontaneous curvature (SC) of the lipid monolayer controls membrane poration, our Poisson model and Boltzmann energetic considerations suggest a transition from a leaky to a non-leaky fusion pathway depending on the SC of the target membrane. When the target membrane SC is below approximately 0.20 nm1 fusion between influenza virus and target membrane is predominantly non-leaky while above that fusion is predominantly leaky, suggesting that influenza hemagglutinin (HA)-catalyzed topological conversion of target membranes during fusion is associated with a loss of membrane integrity. 2. Towards the goal of understanding the pathophysiology of mild blast induced TBI (bTBI), and identifying the physical forces associated with the primary injury phase, we developed a system that couples a pneumatic blast to a microfluidic channel to precisely and reproducibly deliver shear transients to dissociated human central nervous system (CNS) cells, on a time scale comparable to an explosive blast but with minimal pressure transients. Using fluorescent beads, we have characterized the shear transients experienced by the cells, and demonstrate that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell culture volume. This system is compatible with high resolution, time-lapse optical microscopy. Using this system, we demonstrate that blast-like shear transients produced with minimal pressure transients and sub-millisecond rise times activate calcium responses in dissociated human CNS cultures. Cells respond with increased cytosolic free calcium to a threshold shear stress between 8-25 Pa; the propagation of this calcium response is a result of purinergic signaling. We propose that this system models, in vitro, the fundamental injury wave produced by shear forces consequent to blast shock waves passing through density inhomogeneity in human CNS. 3. Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfills critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout, we here demonstrate that the abundant integral PVM protein EXP1 is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase. Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localization of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly loss of EXP1 however affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites. 4. The survival of Plasmodium spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the Plasmodium translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane.
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