Ultrasound-mediated drug delivery is a nonchemical, nonviral, and noninvasive method for targeted transport of drugs and genes into cells. Molecules can be delivered into cells when ultrasound disrupts the cell membrane by a mechanism believed to involve cavitation. This study examined molecular uptake and cell viability in cell suspensions (DU145 prostate cancer and aortic smooth muscle cells) exposed to varying peak negative acoustic pressures (0.6-3.0 MPa), exposure times (120-2000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. With increasing pressure and exposure time, molecular uptake of a marker compound, a calcein, increased and approached equilibrium with the extra cellular solution, while cell viability decreased. Varying pulse length produced no significant effect. All viability and molecular uptake measurements collected over the broad range of ultrasound conditions studied correlated with acoustic energy exposure. This suggests that acoustic energy exposure may be predictive of ultrasound's nonthermal bioeffects.

Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperaturesensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zincbinding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.Sterols are essential structural and regulatory components of eukaryotic cellular membranes (1, 2). However, cholesterol over-accumulation is cytotoxic (3), necessitating mechanisms to maintain this metabolite at appropriate levels. A pivotal component of this homeostasis is the esterification of free sterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) 1 (4, 5).Indeed, the inhibition of ACAT in sterol-loaded cells induces cell death when extracellular sterol acceptors such as high density lipoproteins are absent (6, 7). Intracellular cholesterol redistribution mediates a number of responses to elevated free sterol levels. These include elevated ACAT activity, down-regulated sterol and fatty acid biosynthesis, and reduced lipoprotein uptake via LDL receptors (8, 9). The latter two events reflect changes in transcriptional activation by sterol regulatory element-binding proteins (SREBPs) in response to sterol accumulation in regulatory pools (9), whereas ACAT activity is allosterically regulated by substrate supply (10). Sterols are maintained at a high concentration in the plasma membrane (PM) relative to the endoplasmic reticulum (ER) (1, 2), where SREBP and ACAT reside. Thus trafficking of sterol to and from the ER is a critical component of sterol homeostasis.The process of sterol trafficking is poorly understood at the molecular level. In certain cell types, caveolin influences what has been termed "fast" movement of cholesterol to plasma membrane cholesterol-rich microdomains (caveolae) (11,12). Mutations in the Niemann Pick type C (NPC1) gene result in accumulation of LDL-derived cholesterol in the lysosome (13, 14). However, not all cells express caveolin, and the movement of endogenously synthesized cholesterol to the plasma membrane in NPC1-deficient cells is normal (15).To identify novel genes that mediate sterol trafficking in all higher cells, we utilized the genetically tractable model eukaryote, Saccharomyces cerevisiae (budding yeast). We reasoned that dependence on sterol esterification for viability would be one criterion for identifying novel sterol...

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C–related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.

Ultrasound has been shown to reversibly and irreversibly disrupt membranes of viable cells through a mechanism believed to involve cavitation. Because cavitation is both temporally and spatially heterogeneous, flow cytometry was used to identify and quantify heterogeneity in the effects of ultrasound on molecular uptake and cell viability on a cell-by-cell basis for suspensions of DU145 prostate cancer and aortic smooth muscle cells exposed to varying peak negative acoustic pressures (0.6-3.0 MPa). exposure times (120-2,000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. Cell-to-cell heterogeneity was observed at all conditions studied and was classified into three subpopulations: nominal uptake (NUP), low uptake (LUP), and high uptake (HUP) populations. The average number of molecules within each subpopulation was generally constant: 10(4)-10(5) molecules/cell in NUP, approximately 10(6) molecules/cell in LUP, and approximately 10(7) molecules/cell in HUP. However, the fraction of cells within each subpopulation showed a strong dependence on both acoustic pressure and exposure time. Varying pulse length produced no significant effect. The distribution of cells among the three subpopulations correlated with acoustic energy exposure, which suggests that energy exposure may govern the ability of ultrasound to induce bioeffects by a nonthermal mechanism.

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cWe applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5= rapid amplification of cDNA ends. The ϳ1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection. Murine gammaherpesvirus 68 (MHV68; also known as ␥HV68 or murid herpesvirus 4) infection of mice is a model pathogenesis system for gammaherpesviruses such as Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/ HHV-8) and Epstein-Barr virus (EBV) (5, 20). The life cycles of these lymphotropic and transforming viruses in the host involve the initial transit across a mucosal barrier to gain access to and establish latency in a leukocyte reservoir, followed by intermittent reactivation and dissemination to the mucosal surfaces for spread to new hosts (32,74,77,82).Replication at the site of primary infection impacts MHV68 dissemination and latency establishment in secondary lymphoid tissues of mice. The absence of proteins essential for lytic replication, such as the viral transactivator ORF50/mRTA (61) and the ORF6/single-stranded DNA-binding protein (ssDBP) (49), or the inhibition of viral DNA replication by the administration of cidofovir (62) impairs the establishment of latency in the splenic B cell compartment. In addition, virus replication at the mucosa triggers the innate and adaptive immune responses critical for host control (6,45,72,73). These responses might play a critical role in the recruitment and activation of target cells such as dendritic cells that precede dissemination to distal reservoirs, such as splenic B cells and peritoneal macrophages (5, 25). Thus, the lytic gene expression program in newly infected cells that are permissive for productive infection is an important aspect of pathogenesis.Reactivation from latency is a...

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

Abstract. Increasing adoption of smartphones in recent times has begun to attract more and more malware writers towards these devices. Among the most prominent and widely adopted open source software stacks for smartphones is Android that comes with a strong security infrastructure for mobile devices. However, as with any remote platform, a service provider or device owner needs assurance that the device is in a trustworthy state before releasing sensitive information to it. Trusted Computing provides a mechanism of establishing such an assurance. Through remote attestation, tc allows a service provider or a device owner to determine whether the device is in a trusted state before releasing protected data to or storing private information on the phone. However, existing remote attestation techniques cannot be deployed on Android due to the unique, vm-based architecture of the software stack. In this paper, we present an attestation mechanism tailored specifically for Android that can measure the integrity of a device at two levels of granularity. Our approach allows a challenger to verify the integrity of Android not only at the operating system level but also that of code executing on top of the vm. We present the implementation details of our architecture and show through evaluation that our architecture is feasible both in terms of time complexity and battery consumption.