Real-time imaging of plasma membrane dynamics in sonoporation: from perforation to recovery

The Conference program & abstracts' website is located at http://www.ewh.ieee.org/conf/uffc/2013/Session IUS1-K3: Metrology and sonoporation: abstract no. IUS1-K3-4BACKGROUND, MOTIVATION AND OBJECTIVE: To properly harness sonoporation for therapeutic applications, it is unarguably vital to characterize the fundamental biophysical processes involved. Of particular relevance are two membrane-level processes that epitomize the notion of sonoporation: 1) how membrane perforation is induced by ultrasound-microbubble interactions, and 2) how the membrane remodels itself following an episode of sonoporation. Acquiring direct observations of these processes is however not a straightforward task (ironically, these membrane-level events have yet to be convincingly demonstrated in-situ). In this investigation, our aim is to acquire the first series of direct evidence on the time course of membrane perforation and recovery in sonoporation. In particular, we seek to unravel the time-varying surface topography of sonoporated cell membrane in-situ. STATEMENT OF CONTRIBUTION/METHODS: A real-time imaging platform for monitoring of cell-microbubble interactions was first developed, and it was a composite system that coupled a 1 MHz ultrasound module to a laser scanning confocal microscope. A nose-cone shaped waveguide (1” diameter, 7.5 cm height) was devised to align the ultrasound beam focus to the microscope’s imaging plane. This waveguide was angled at 45 deg. with respect …postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

Ultrasound as a repulsive cue for neuronal development: real-time morphological observations in-vitro

Session IUS1-PA4: Bioeffects and metrology: abstract no. IUS1-PA4-6The Conference program & abstracts' website is located at http://www.ewh.ieee.org/conf/uffc/2013/BACKGROUND, MOTIVATION AND OBJECTIVE: A core event in neuronal development is the outgrowth of neurite branches from a neuron’s cell body. It is known that, by presenting physical or chemical cues to the extracellular environment, neuronal development can potentially be modulated. In this work, we seek to investigate the use of ultrasound in modifying neuronal development behavior. We hypothesize that ultrasound, when applied at low intensity and pulsed settings, can possibly serve as a physical cue that operates like mechanical stimulation. STATEMENT OF CONTRIBUTION/METHODS: Our study was carried out on a customized platform that allows real-time imaging of neuronal morphology. Pulsed ultrasound (1 MHz frequency, 20 or 100 cycle pulse lengths, 500 or 2500 Hz PRF) was applied through a waveguide at a 45 degree angle with respect to the microscope’s field of view, which is aligned to the center of a 100mm polystyrene cell dish. Three different peak acoustic pressures (measured in-situ) were used: 0.13, 0.40, 0.84 MPa (correspond to SPTA intensities of up to 1.168 W/cm2 for our given pulsing parameters). In this work, N2a mouse neuroblastoma was used as the …postprin

Cytoplasmic signaling involved in sonoporation-induced apoptosis andmitosis repression of myeloid leukemia cells

Sonoporation is known to be a transient phenomenon that may disrupt thehomeostasis of living cells. In this work, we showed that sonoporation may beartime-lapse impact on cellular viability through up-regulation of cytoplasmicsignaling proteins related to apoptosis and cell-cycle arrest. Our experimentswere done on HL-60 leukemia cells (10 6 cells/ml), and sonoporationwas induced via the use of 1% v/v microbubbles and 1-min. pulsed ultrasoundexposure (0.5MPa peak negative pressure, 1MHz center frequency, 10% duty cycle,1 kHz pulse repetition frequency). The transient nature of sonoporation in thesecells was confirmed by performing scanning electron microscopy on selected cellsamples that were fixed respectively after a few seconds into the ultrasoundexposure and one minute after the end of exposure. Cytoplasmic signaling changesof these cells were studied at four post-sonoporation time points (4h, 8h, 12h,24h) using western blot analysis. Five signaling proteins related to apoptosisand mitosis were analyzed in this work: 1) PARP (for DNA repair); 2)cleaved-PARP (fragments due to cleavage by pro-apoptotic caspase proteins); 3)Bcl-2 (inhibitor for mitochrondrial release of pro-apoptotic molecules); 4) Bax(complement of Bcl-2); 5) Cdc-2 (regulator for cell mitosis). Three key resultswere found from the cytoplasmic signaling analysis. First, PARP levels werereduced over the monitoring period whilst cleaved-PARP had increased inexpression, and in turn they indicate that the cells' anti-apoptotic responseswere dampened following sonoporation and pro-apoptotic caspase proteins werelikely activated. Second, drop in Bcl-2 and rise in Bax were observed, and thesesuggest that the mitochondrion was involved in apoptotic signal transductioninside sonoporated cells. Third, Cdc-2 was seen to decrease, implying thatmitosis was repressed in sonoporated cells. © 2010 IEEE.published_or_final_versionThe 2010 IEEE International Ultrasonics Symposium, San Diego, CA., 11-14 October 2010. In Proceedings of IEEE IUS, 2010, p. 1771-177

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study

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Immunosuppressive compounds of Pestalotiopsis sp., an endophytic fungus of Tripterygium wilfordii

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Protection of lethal toxicity of endotoxin by Salvia miltiorrhiza BUNGE is via reduction in tumor necrosis factor alpha release and liver injury

Lipopolysaccharide (LPS) has been implicated as one of the major cause of Gram-negative bacteria-induced sepsis that are life-threatening syndromes occurring in intensive care unit patients. Many natural products derived from medicinal plants may contain therapeutic values on protecting endotoxemia-induced sepsis by virtue their ability to modulate multiple pro-inflammatory cytokines. In the present study, we show that Salvia miltiorrhiza (SM) BUNGE or Danshen, used in treatment of various systemic and surgical infections in the hospitals of China, was able to block the lethal toxicity of LPS in mice via suppression of TNF-α release and protection on liver injury. The ability of SM to suppress LPS-induced TNF-α release is further confirmed by in vitro experiments conducted on human peripheral blood leukocytes (PBL) and the RAW 264.7 macrophage cell line. Immunophenotyping by flow cytometry shows improved T-helper cell (CD4) and T-suppressor cells (CD8) ratio in SM-treated PBL and splenocytes of LPS-challenged mice. The drop in plasma glutamate-pyruvate transaminase (GPT) induced by LPS provides evidence that SM can protect hepatic damage. The present study explains some known biological activities of SM, and supports the clinical application of SM in the prevention of inflammatory diseases induced by Gram-negative bacteria. © 2005 Elsevier B.V. All rights reserved.postprin

Sonoporation-induced endoplasmic reticulum stress: signaling pathway analysis

Session IUS1-K3: Metrology and sonoporation: abstract no. IUS1-K3-6The Conference program & abstracts' website is located at http://www.ewh.ieee.org/conf/uffc/2013/BACKGROUND, MOTIVATION AND OBJECTIVE: Sonoporation can induce a complex range of biological consequences beyond a transient porosity change at the membrane level. In particular, at a subcellular level, the functioning of various organelles may become disrupted. This work represents the first demonstration that the endoplasmic reticulum (ER) (an organelle responsible for protein folding, lipid synthesis, and calcium regulation) may be actively involved in mediating post-sonoporation cellular response. STATEMENT OF CONTRIBUTION/METHODS: We have assayed for various ER-related proteins using Western blot analysis. HL-60 cell suspensions were used …postprin