e. what was the landscape of the central lagoon before the first human settlements, what were the consequences of the major river diversions and what were the consequences of dredging new navigation channels during the last century? First, we found that the landscape of the central lagoon (between the city of Venice and the main land) before the first human settlements went through different phases: during the Holocene before the lagoon ingression, this area was an alluvial plain belonging to the Brenta megafan close to the internal margin of the lagoon. In this period a river channel

(CL2), probably a channel of the Brenta river, crossed the coastal plain in the Eneolithic and Bronze ABT-888 order Age, when the first demographic boom occurred in the area. The lagoon environment foraminifera found in the channel sands testify the tidal influence and the proximity of the river mouth to the lagoon. Furthermore, the presence of a salt marsh and of a tidal channel

(CL1) in the western part of the study area dating back to around 800 BC is evidence of the lagoon expansion in the Iron Age, before the first stable human settlements in the lagoon. During this expansion, the river channel CL2 got gradually more brackish properties until it became a tidal channel called “Canale di Bottenigo” flowing into the Giudecca Channel, one of the main channels in the historical center of the city of Venice. Second, as a consequence of the artificial diversion of major rivers many channels disappeared in the area. In particular, because of the closure of the

Brenta river this website mouth in the 12th century, no longer active channel CL2 was filled by mudflat lagoonal sediments. Third, the comparison with historical maps starting from 1691 AD shows a general simplification of the morphologies over the centuries Glycogen branching enzyme with a drastic reduction of the number of channels. After the dredging of the main industrial and navigation channels, we observe an acceleration of this morphological simplification in the last century, with the filling up of many natural channels. The reconstruction of the “Coa de Botenigo” (CL3) shows an example of this process: as a consequence of the Vittorio Emanuele III Channel dredging, the meanders of the CL3 palaeochannel and their ramifications completely disappeared. These results may indicate that a new dredging of a large navigation channel in the area, by inducing a higher energetic hydrodynamic regime, could increase the filling up of the channels and accelerate the ongoing deepening trend in the area as happened in the lagoon of Aveiro in Portugal. As is shown in this case study, the advance of engineering technology in the last few centuries increased the tendency to ‘freeze’ the coastal lagoons by creating ‘fixed’ structures (fixed inlets, harbors, new dredged channels, barriers, etc.).

i.d., 5 μm, Torrance, CA, USA) were used for HPLC analysis. MicroTOF-Q II LC/MS (Bruker Daltonics, Bremen, Germany) was used for the LC/MS analysis. A549 lung cancer cells line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). DMEM/F12 media, fetal bovine serum, penicillin/streptomycin antibiotics, and phosphate buffer saline (PBS) were purchased from

Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Autophagy Compound Library purchase bromide (MTT) was purchase from Amresco (Solon, OH, USA), and 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH), DMSO were purchased from Sigma Aldrich (St. Louis, MO, USA). SpectraMax 340PC384 microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used to measure the absorbance of the samples. HPLC solvents and other reagents were purchased from Duksan (Ansan, Korea). Ginsenoside standards were isolated and identified from KG and VG in our laboratory [2] and [12]. Dried VG, including radix, rhizome, and hairy root, was ground and sieved to get the powder of 355–425 μm. A 150 mg portion of each powdered VG sample was put into stainless steel vessel with 1.5 mL

of distilled water. The vessel was closed tightly and PS-341 in vivo heated in an oven for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, or 20 h at 120°C. After heating, the samples were lyophilized to yield a dried powder, which were extracted three times by ultrasonication at 65°C for 3 h, 1.5 h, and 1 h, using 10 mL, 10 mL, and 5 mL of methanol (MeOH), respectively. The combined extract was centrifuged and then made up to 25 mL with MeOH. A 2 mL of the MeOH extract of each sample was dried under nitrogen stream. The residue was dissolved in 1 mL of MeOH and then filtered through a 0.45 μm membrane filter prior to HPLC analysis. The MeOH extract of each sample was dried under

nitrogen stream, then dissolved in DMEM/F12 media containing 0.1% DMSO to get various concentrations for the cell proliferation analysis. The MeOH extract of each sample was used at the final concentration Calpain equivalent to 6 mg of dried VG powder in 1 mL of MeOH. The reported method [15] was applied for the HPLC analysis of ginsenosides with a slight modification. Separation was achieved by using Phenomenex C18 column (250 mm × 4.6 mm. i.d., 5 μm) and the following gradient program with 5% acetonitrile (A) and 95% acetonitrile (B): 0–20 min (85–80% A); 20–45 min (80–52.5% A); 45–55 min (52.5–0% A); 55–65 min (0% A). Flow rate was set at 1 mL/min and injection volume was 20 μL. ELSD was set to a probe temperature of 80°C, and nebulizer gas (N2) flow was adjusted to 1.5 L/min. A549 lung cancer cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% antibiotics in a humidified atmosphere of 5% CO2 at 37°C. Antiproliferative activity was measured by a previously reported method [16]. A549 lung cancer cells at 104 cells/well were seeded in 96-well plates and incubated for 24 h.

, 2007), and atherosclerosis ( Arunachalam et al., 2010). All these factors promote progressive blood flow restriction to pulmonary vascular bed, leading to right ventricular hypertrophy. Selleckchem Autophagy inhibitor Huh and colleagues have reported that BMDMCs alleviate pulmonary hypertension in a cigarette smoke-induced emphysema model, inhibiting muscularization

in small pulmonary vessels and stimulating VEGF-induced angiogenesis ( Huh et al., 2011). Similarly, in the current study, a right cardiac dysfunction was also detected in E-SAL, which was significantly minimized in the E-CELL group. This behavior was accompanied by a marked reduction in collagen fiber content in airways, pulmonary wall vessels, and alveolar septa, and associated with a lower mRNA expression of TGF-β and PDGF. Severe COPD leads to cor pulmonale combined with secondary reduction in left ventricular filling, stroke volume

and cardiac output ( Barr et al., 2010). Nevertheless, no left ventricular dysfunction was found in our study, which implies that the present murine Selleck Everolimus model of elastase-induced emphysema did not reach such high severity and/or did not have sufficient time to develop. The present study has some limitations: (1) BMDMC were injected 3 h after first elastase administration. Consequently, more studies should be performed to analyze BMDMC effects after the injury is established; (2) all data were analyzed at 5 weeks. Therefore, the time course analysis following BMDMC therapy was not performed, limiting the understanding of the early effects of cell therapy; (3) Y chromosome DNA was also studied only at 5 weeks in cell-treated groups, and the behavior of BMDMC immediately after injection was not analyzed; (4) elastolysis

was not evaluated using casein and elastin zymography but electron microscopy, and (5) we were not able to determine whether BMDMC had a direct beneficial effect on the heart or an indirect benefit mediated by improvement of lung injury. Therefore, future studies analyzing heart data, such as right ventricular weight, collagen fiber content, apoptosis, and cytokine/growth factor expressions will be required SPTLC1 to better elucidate the direct effect of elastase or cell therapy on the heart. In conclusion, in the present murine model of pulmonary elastase-induced emphysema, BMDMC therapy was effective to prevent lung and cardiovascular damage. These beneficial effects might be attributed to paracrine effects modulating the expression of growth factors involved in the pathogenesis of emphysema. The authors would like to express their gratitude to Mr. Andre Benedito da Silva for animal care, Miss Thaiana Borges de Sousa for her skilful technical assistance during the experiments, Mrs. Ana Lucia Neves da Silva for her help with microscopy, and Ms. Claudia Buchweitz and Mrs. Moira Elizabeth Schöttler for their assistance in editing the manuscript.

For non-scalar expressions, the distribution was 12, 2 and 6 respectively. This classification reveals that the majority (17 and 18

out of 20 children for scalars and non-scalars see more respectively) were consistent in their behaviour (either informative or underinformative). This finding is in line with the participant distributions reported by Guasti et al. (2005) for children and Bott and Noveck (2004) for adults for the scalar expressions. It further justifies the conclusion that many children lack some aspect of pragmatic competence important to performing this task. Not only was there a difference at the group level between the rejection of underinformative and false utterances, but at the individual level the majority of children (13 out of 20 for scalars and 12 out of 20 for non-scalars) consistently accepted underinformative utterances. As mentioned, many adult responses did not consist of a straightforward acceptance or rejection, but were more indirect, phrased as revisions or meta-linguistic remarks. Indirect responses were obtained in the underinformative condition only, at rates of 12% Entinostat in vitro and 33% for scalars and non-scalars respectively (as a proportion

of all non-acceptances). More than 90% of these indirect responses were revisions starting with ‘yes’, ‘true’ or ‘right’, followed by the informative description (either with the use of ‘but’ or ‘and’ or without any conjunction). For instance, one adult participant said “yes, he picked up all of them”, and “yes, but he also painted the heart”. The remaining indirect responses did not commit with regard to the correct binary value of the utterance (‘right’ or ‘wrong’) but included explicitly meta-linguistic remarks such as “half right, half wrong”, “I can’t really tell”,

“I don’t know”. If the indirect responses are scored as incorrect, then adult performance in the underinformative check details conditions falls to 88% for scalars and 67% for non-scalars. Adults are still outperforming the children for both types of expression (Mann–Whitney U: both U > 3.03, p < .001, r > .47), but there is a main effect of expression, with the adults performing higher with scalars than with non-scalars (Wilcoxon Signed Ranks test, W = 2.03, p < .05, r = .45). The presence of indirect responses in the underinformative but not in the logically false condition indicates that adults do not consider violations of informativeness to be as grave as violations of logical truth. However, no other study using a similar paradigm (e.g. Guasti et al., 2005, experiment 4; Papafragou & Musolino, 2003, both experiments) reports any indirect responses from adults. Could this mean that there is something erroneous with the task that we designed? We think this unlikely on two grounds.

Or do they? In this paper, I argue that in fact many of us mistake landscapes altered by humans in the past for wilderness that has never experienced substantial human influences, and that this misperception hampers our ability to understand the intensity and extent of human manipulation of Earth surfaces. By more fully comprehending the global implications of human manipulations during the Anthropocene,

we can more effectively design management to protect and restore desired landscape and ecosystem qualities. This is a perspective paper rather than a presentation of new research results. I write from the perspective of a geomorphologist, but much of what I describe below applies to anyone who studies the critical zone – Earth’s near-surface layer from the tops of the trees down to the deepest learn more groundwater – and who wishes to use knowledge of critical zone processes and history to manage landscapes

and ecosystems. I use landscape to refer to the physical configuration of the surface and near-surface – topographic relief, arrangement of river networks, and so forth – and the fluxes that maintain physical configuration. I use ecosystem to refer to the biotic and non-biotic components and processes of a region. In practice, the two entities are closely intertwined because the landscape creates habitat and resources for the biota and biotic activities shape the landscape. I distinguish the two entities only because the time scales over which each changes can differ and the changes may not be synchronous. The Branched chain aminotransferase title of this paper alludes to the find more now well-known paper, “Stationarity is dead: whither water management?” (Milly et al., 2008). I use the phrase “wilderness is dead” because I interpret wilderness in the strictest sense, as a region that people have never influenced. Given warming climate and rapidly melting glaciers and sea ice, even the most sparsely populated polar regions no longer qualify as wilderness under this interpretation.

Just as stationarity in hydrologic parameters has ceased to exist in an era of changing climate and land use, so has wilderness. I use this realization to explore the implications of the loss of wilderness for critical zone studies and management from the perspective of a geomorphologist. I start by briefly reviewing the evidence for extensive human alteration of the critical zone. I explore the implications for geomorphology of a long history of widespread human alteration of the critical zone in the context of three factors of interest to geomorphologists (historical range of variability, fluxes of matter and energy, and integrity and sustainability of critical zone environments). I then explore how concepts of connectivity, inequality, and thresholds can be used to characterize critical zone integrity and sustainability in specific settings.

Our results demonstrate that chronic alcohol feeding results in a decrease in AMPK activity, which is recovered by RGE treatment. Previously, we reported that feeding mice with a Lieber–DeCarli diet containing 5% EtOH for 10 days, followed by a single dose of EtOH gavage (5 g/kg body weight) (chronic–binge EtOH model) induces significant fatty liver and liver injury

with oxidative stress (Fig. 6A) [25]. To investigate the effect of RGE for the treatment of Idelalisib ALD using the chronic–binge EtOH model, EtOH-fed mice were treated with RGE. Treatment with RGE decreased EtOH-induced serum ALT and AST levels (Fig. 6B). The protective effect of RGE on alcoholic steatosis was further confirmed by liver histology as shown by H&E staining. It was noted that treatment of alcohol-fed mice with RGE completely inhibited fat infiltration (Fig. 6C), confirming selleck chemicals the ability of RGE to inhibit fat accumulation in liver. Moreover, the chronic–binge EtOH model significantly increased 4-HNE positive cells, which is consistent with our previous report [25]. However, similar to the chronic EtOH model, the amount of 4-HNE positive cells was dose-dependently and significantly reduced by treatment with RGE (Fig. 7A). RGE also markedly attenuated nitrotyrosine positive cells, confirming that RGE is capable of inhibiting alcohol-induced oxidative stress in the chronic–binge EtOH animal model (Fig. 7B). We next examined the effect of RGE on

fat accumulation in a mouse hepatocyte cell line, AML12. EtOH treatment for 3 days increased fat accumulation in hepatocytes as Montelukast Sodium shown by Oil red O staining. However, RGE (500 μg/mL or 1000 μg/mL) treatment reduced fat accumulation in a dose-dependent manner (Fig. 8A). To determine whether changes of fat accumulation in the hepatocyte were consistent with lipogenesis- or lipolytic-associated gene expression, the expression of SREBP-1, Sirt1, and PPARα was observed by Western blot analysis following concomitant treatment with 10–1000 μg/mL of RGE and EtOH for 3 days. In agreement with the in vivo data, RGE inhibited the ability of EtOH to induce SREBP-1 and repress Sirt1

and PPARα expression in AML12 cells ( Fig. 8B). The pharmacological properties of ginseng are primarily attributed to a group of active ingredients, the ginsenosides, which are a diverse group of steroidal saponins. Gum and Cho recently reported that total ginsenoside amount of RGE was 19.66 mg/g containing the major ginsenosides Rb1 (4.62 mg/g), Rb2 (1.83 mg/g), Rc (2.41 mg/g), Rd (0.89 mg/g), Re (0.93 mg/g), Rf (1.21 mg/g), Rg1 (0.71 mg/g), Rg2 (3.21 mg/g), Rg3 (3.05 mg/g), Rh1 (0.78 mg/g), and other minor ginsenosides [21]. Therefore, we next identified the major component of red ginseng required for the inhibition of hepatic steatosis. We determined the effects of the major ginsenosides Rb1, Rb2, and Rd on the EtOH-induced fat accumulation in AML12 cells.

The mice were given free access to control diet or alcohol Lieber–DeCarli liquid selleck chemicals diet for 4 weeks with or without RGE (250 mg/kg or 500 mg/kg, per os, n = 8) The mice were randomly assigned to the groups specified. The second was a mouse model of chronic–binge EtOH intake. The mice were fed with the control diet for 5 days, and then divided into four groups. The EtOH groups were fed with the Lieber–DeCarli liquid diet containing 5% EtOH for 10 days with or without RGE (250 mg/kg or 500 mg/kg, per os, n = 8). The control groups were pair-fed the

control diet for 10 days. At Day 11, mice in EtOH groups were gavaged a single dose of EtOH (5 g/kg body weight, 20% EtOH), whereas mice in control groups were gavaged isocaloric dextrin maltose. The mice were sacrificed 9 hours after gavage. AML12 cell lines were purchased from ATCC (Manassas, VA, USA). Cells were plated at a density of 3 × 105/well in 60 mm dishes and grown to 70–80% confluency. Cells were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 containing 10% fetal bovine serum (Hyclone, Logan, UT, USA), 50 units/mL penicillin, 50 μg/mL streptomycin, Selleckchem Osimertinib 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone at 37°C in a humidified atmosphere with 5% CO2. RGE or ginsenosides were dissolved in phosphate-buffered saline (PBS) and added to the cells. The cells were then incubated at

37°C for the indicated time period, and washed twice with ice-cold PBS prior to sample preparation. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase Teicoplanin (AST) were analyzed using Spectrum, an automatic blood chemistry analyzer (Abbott Laboratories, Abbott Park, IL, USA). Samples from the liver

were separated and fixed in 10% neutral buffered formalin. The samples were then embedded in paraffin, sectioned (3–4 μm), and stained with hematoxylin and eosin (H&E) for general histopathological analysis. In addition, the effect of RGE treatment on the 4-HNE and nitrotyrosine immunoreactivity was also observed by immunohistochemical methods. For the analysis of fat accumulation in the liver, 10-μm sections were cut from frozen samples and stained with Oil Red O for 10 min. The slides were rinsed in water and counterstained with Mayer’s hematoxylin, followed by analysis using light microscopy. Lipid droplet formation in hepatocytes was determined by Oil Red O staining. Cells were grown on a six-well plate. After treatment, the cells were fixed 4% formaldehyde in PBS for 1 h and rinsed with 60% isopropanol. Cells were then stained with Oil Red O solution. Hepatic lipid content was measured as described previously [25]. Briefly, lipids from the total liver homogenate were extracted using chloroform/methanol (2:1), evaporated, and dissolved in 5% triton X-100. Triglyceride content was determined using Sigma Diagnostic Triglyceride Reagents (Sigma).

, 2009) and was supported by both the quasi-stable sea level in the Black Sea since the mid Holocene (Giosan et al., 2006a and Giosan et al., 2006b) and the drastic increase in discharge over the last 1000–2000 years (Giosan et al., RO4929097 purchase 2012). Second, delta fringe depocenters supporting delta lobe development are associated only with the mouths of major distributaries, but their volume is influenced by both sediment discharge and mouth morphodynamics. Lobes develop and are maintained not only via repartitioning most of the sediment

load to a single distributary but also by trapping of fluvial and marine sediments at the wave-dominated mouths of small discharge distributaries and periodically releasing them downcoast (Giosan et al., 2005). In this way, multiple lobes with different morphologies can coexist, abandonment of wave-dominated lobes is delayed and, by extension, the intensity BMS-754807 cell line of coastal erosion is minimized. River delta restoration as defined by Paola et al. (2011) “involves diverting sediment and water from major channels into adjoining drowned areas, where the sediment can build new land and provide

a platform for regenerating wetland ecosystems.” Such strategies are being currently discussed for partial restoration of the Mississippi delta, because the fluvial sediment load there is already lower than what is necessary to offset the already lost land ( Turner, 1997, Blum and Roberts, 2009 and Blum and Roberts, 2012). The decline in fluvial sediment load on the Mississippi cAMP combined with the isolation of the delta plain by artificial levees and enhanced subsidence have led to enormous losses of wetland, but capture of some fluvial sediment that is now lost at sea (e.g., Falcini et al., 2012) is envisioned via controlled river releases during floods and/or diversions

( Day et al., 1995, Day et al., 2009, Day et al., 2012 and Nittrouer et al., 2012). Strategies are designed to maximize the capture of bedload, which is the primary material for new land build up ( Allison and Meselhe, 2010 and Nittrouer et al., 2012) and they include deep outlet channels and diversions after meander bends where lift-off of bed sand increases. Mass balance modeling for the Mississippi delta indicates that between a fourth and a half of the estimated land loss could be counteracted by capturing the available fluvial sediment load ( Kim et al., 2009). Sand is indeed needed to nucleate new land in submerged environments, but enhancing the input of fine sediments to deltaic wetlands should in principle be an efficient way to maintain the delta plain that is largely above sea level because fine suspended sediments make up the great bulk of the sediment load in large rivers (e.g., 98–95%; Milliman and Farnsworth, 2011).

, 2011, Steffen et al., 2011, Zalasiewicz et al., 2011a and Zalasiewicz

et al., 2011b). Rather GDC-0449 in vitro than constituting a formal chronostratatgraphic definition of the Anthropocene epoch, this consensus adopts, as a practical measure, a beginning date in the past 50–250 years: In this paper, we put forward the case for formally recognizing the Anthropocene as a new epoch in Earth history, arguing that the advent of the Industrial Revolution around 1800 provides a logical start date for the new epoch. (Steffen et al., 2011, p. 842) Steffen et al. (2011) follow the lead of Crutzen and Stoermer (2000) in identifying the rapid and substantial global increase in greenhouse gasses associated with the Industrial Revolution as marking the onset of the Anthropocene, while also documenting a wide range of other rapid increases in human activity since 1750, from the growth of McDonald’s restaurants to expanded

fertilizer use (Steffen et al., 2011, p. 851). In identifying massive and rapid evidence for human impact on the earth’s atmosphere as necessary for defining the Holocene–Anthropocene transition, and requiring such impact to be global in scale, Steffen et al. (2011) are guided by the formal criteria employed by the International Commission on Stratigraphy (ICS) in designating geological time learn more units. Such formal geologic criteria also play a central role the analysis of Zalasiewicz et al. (2011b) in their comprehensive consideration of potential and observed stratigraphic markers of the Anthropocene: “Thus, if the Anthropocene is to take it’s http://www.selleck.co.jp/products/Docetaxel(Taxotere).html place alongside other temporal divisions of the Phanerozoic, it should be expressed in the rock record with unequivocal and characteristic stratigraphic signals.” (Zalasiewicz et al., 2011b, p.

1038). Ellis et al. (2011) also looks for rapid and massive change on a global scale of assessment in his consideration of human transformation of the terrestrial biosphere over the past 8000 years, and employs a standard of “intense novel anthropogenic changes …across at least 20 per cent of Earth’s ice-free land surface” as his criteria for “delimiting the threshold between the wild biosphere of the Holocene and the anthropogenic biosphere of the Anthropocene” (2011, p. 1027). A quite different, and we think worthwhile, approach to defining the onset of an Anthropocene epoch avoids focusing exclusively and narrowly on when human alteration of the earth systems reached “levels of equal consequence to that of past biospheric changes that have justified major divisions of geological time” (Ellis, 2011, p. 1027). We argue that the focus should be on cause rather than effect, on human behavior: “the driving force for the component global change” (Zalasiewicz et al., 2011a, p.

Such strategies have to be developed for the new two-step nanoprecipitation procedure as well. Having accomplished protein loaded nano-sized PLGA particles, we tested the development of the sustained release nanoparticles into an application platform. We selected Cyt-c as

model protein because it has been employed in experiments geared towards better cancer treatment options [24]. The size of our particles makes them potentially useful in passive and also active targeting of cancer tissues [37,38]. For example, Santra et al. [24] demonstrated recently the therapeutic potential of Cyt-c in nanoparticles by their capability to induce apoptosis in lung carcinoma cells after uptake by the cells by endocytosis. However, their vehicle consisted of a water-soluble hyperbranched Pexidartinib cost polyhydroxyl polymer not JQ1 research buy approved in medical applications. In contrast, our nanoparticles employ an already FDA approved and commercially available polymer (PLGA) and a straight forward encapsulation method. We hypothesized that encapsulation of Cyt-c via the two step nanoprecipitation method should work using the optimum conditions identified for lysozyme (Table 7) because both proteins have a similar molecular weight (12 and 14▒kDa, respectively) and are basic [39]. The encapsulation efficiency for Cyt-c was with 72% is similar to that obtained for lysozyme under identical conditions (Table 8). The peroxidase activity of

Cyt-c was comparable to values prior to precipitation and encapsulation and only few aggregates were formed indicating good preservation of structural integrity during the process. The size of the

particles obtained was 340▒nm and thus potentially useful to enable passive delivery to cancer tissues based on the EPR effect [37,38]. In vitro release of Cyt-c from the PLGA nanoparticles showed an initial “burst” release within 24▒h that was reasonably small with ca. 20% ( Fig. 2). Burst release values of >20% are frequently found for such Tau-protein kinase systems, in particular when nanosized systems are being used [ 40]. During a 100-day incubation period, Cyt c was released completely from the nanospheres. Since the release was slow, the amount of protein released per day was small and the residual activity during release could not be measured with accuracy. Future experiments using cell cultures and animal models will shed light into the bioactivity of the developed system. However, since 100% of the protein was released, we can exclude the formation of buffer-insoluble Cyt-c during the release period. Since there are some reports that PLGA nanoparticles could be internalized by cells, we investigated whether the Cyt-c-PLGA NPs would be toxic to cancer cells. We selected a human cervical cancer cell line (HeLa) as a model system and incubated the cells for 24, 48, 72, and 96▒h at 37▒°C under 5% CO2 with various concentrations of drug-loaded and empty PLGA nanoparticles and determined the cell viability (Fig. 3).