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Hypoxia is considered an important factor in the proliferation and stemness of mesenchymal stem cells (MSCs). This study aimed to obtain large numbers of mouse clonal MSCs (mc-MSCs) and evaluate the effect of hypoxia on the proliferation and differentiation of mc-MSCs. mc-MSCs were cultured on a Cytodex 3 microcarrier in a spinner flask for a suspension culture to increase mass productivity. To produce the hypoxic conditions, CoCl2 and Na2SO3 were used in a low glucose DMEM medium. The hypoxia environment was established using 0.5g/L Na2SO3 with 10μM or 100 µM CoCl2 for 24 hours. As a result, the proliferation of mc-MSCs under hypoxic conditions was 1.56 times faster than the control group over seven days. The gene expression of HIF-1a and VEGFA increased 4.62 fold and 2.07 fold, respectively. Furthermore, the gene expression of ALP, RUNX2, COL1A, and osteocalcin increased significantly by 9.55, 1.55, 2.29, and 2.53 times, respectively. In contrast, the expression of adipogenic differentiation markers, such as PPAR-γ and FABP4, decreased. These results show that the hypoxia environment produced by these chemicals in a suspension culture increases the proliferation of mc-MSCs and promotes the osteogenic differentiation of mc-MSCs.

Although immunotherapy holds promising cytotoxic activity against lymphoma or leukemia, the immunosuppressive mechanisms of solid tumors remain challenging. In this study, we developed and applied a hypergravity exposure system as a novel strategy to improve the responsiveness of breast cancer cells to natural killer (NK) cells for efficient immunotherapy. Following exposure to hypergravity, either in the presence or absence of NK cells, we investigated for changes in the cell cytoskeletal structure, which is related to the F-actin mediated immune evasion mechanism (referred to as “actin response”) of cancer cells. Breast cancer cell line MDA-MB-231 cells were exposed thrice to a 20 min hypergravitational condition (10 × g), with a 20 min rest period between each exposure. The applied hypergravity induces changes in the intracellular cytoskeleton structure without decreasing the cell viability but increasing the cytotoxicity of MDA-MB-231 from 4 to 18% (4.5-fold) at a 3:1 ratio (NK-to-target). Analyses related to F-actin further demonstrate that the applied hypergravity results in rearrangement of the cytoskeleton, leading to inhibition of the actin response of MDA-MB-231. Taken together, our results suggest that the mechanical load increases through application of hypergravity, which potentially improves efficiency of cell-based immunotherapies by sensitizing tumors to immune cell-mediated cytotoxicity.

Local anesthesia is a drug that penetrates the nerve cell membrane and binds to the voltage gate sodium channel, inhibiting the membrane potential and neurotransmission. It is mainly used in clinical uses to address the pain of surgical procedures in the local area. Local anesthetics (LAs), however, can be incorporated into the membrane, reducing the thermal stability of the membrane as well as altering membrane properties such as fluidity, permeability, and lipid packing order. The effects of LAs on the membrane are not yet fully understood, despite a number of previous studies. In particular, it is necessary to analyze which is the more dominant factor, the membrane affinity or the structural perturbation of the membrane. To analyze the effects of LAs on the cell membrane and compare the results with those from model membranes, morphological analysis and 50% inhibitory concentration (IC50) measurement of CCD-1064sk (fibroblast, human skin) membranes were carried out for lidocaine (LDC) and tetracaine (TTC), the most popular LAs in clinical use. Furthermore, the membrane affinity of the LAs was quantitatively analyzed using a colorimetric polydiacetylene assay, where the color shift represents their distribution in the membrane. Further, to confirm the membrane affinity and structural effects of the membranes, we performed an electrophysiological study using a model protein (gramicidin A, gA) and measured the channel lifetime of the model protein on the free-standing lipid bilayer according to the concentration of each LA. Our results show that when LAs interact with cell membranes, membrane affinity is a more dominant factor than steric or conformational effects of the membrane.

Recently, the importance of inhalation toxicity assessment increased due to recent humidifier disinfectant-associated deaths in children. Benzalkonium chloride (BAC) is currently used as a cationic surfactant and germicide in food industry processing lines and as a hand sanitizer. Animal models are mainly used as a method of evaluating the inhalation toxicity of a hazardous substance, but that approach requires considerable amounts of time and cost. As a replacement for animal experiments, in vitro cell culture can be used to assess toxicity. However, such culture does not reflect the natural microenvironment of the lung, particularly its dynamic nature. In this study, we simulated normal breathing levels (tidal volume 10%, 0.2 Hz) through surface elongation of an elastic membrane in a dynamic culture system. The low-cost dynamic system provided easy control of breathing rate during lung cell culture. We assessed the toxicity using different concentrations of BAC (0, 2, 5, 10, 20, and 40 μg/mL) under static and dynamic culture conditions. Following 24 h of exposure to BAC, cellular metabolic activity, cell membrane integrity, interleukin-8 (IL-8) and reactive oxygen species (ROS) levels, and the total amount of protein in cells were analyzed. Our results showed that significant differences in cellular metabolic activity, as well as IL-8 and ROS profiles, between static and dynamic cell growth conditions, following BAC exposure.

Diabetes mellitus is a chronic, progressive disease that has lasting effects on several organs and tissues. In addition, high glucose levels, high triglyceride and high cholesterol levels increase patient health problems. In this study, we investigated natural materials that can alleviate diabetes symptoms or prevent its onset and assessed extracts of bamboo stem, which has been known for centuries as a functional food. Mouse 3T3-L1 cells were induced to differentiate by adding a mixture of dexamethasone, 3-isobutyl-1-methylxanthine, and insulin (DMI) to the cell media after cells reached 100% confluence. The cells were then treated with hot water extract (HE), ethanol extract (EE), or methanol extract (ME) of bamboo stem for 4 and 13 days, after which the expression levels of differentiation markers C/EBPβ, PPARγ, and FABP4 were measured. At day 13, changes in metabolic function were assessed by analyzing the expression levels of ABCG1, CD36, and GLUT4 using RT-qPCR. Adipocyte size distribution was determined using a Coulter counter after trypsinization. The total triglyceride content was quantified using an adipogenesis assay kit. The HE-, EE-, and ME-treated 3T3-L1 cells showed increased adipogenesis marker expression, triglyceride content, and adipocyte volumes compared to the controls. The expression of genes related to adipocyte function were elevated in EE- and ME-treated samples. Taken together, these findings indicate that bamboo stem extracts, especially ethanol extract, have prophylactic effects for diabetes that occur through the promotion of adipogenesis and adipocyte functions.

Globally, approximately a billion patients are estimated to suffer from neurological disorders. Although there are many therapeutic candidates for the central nervous system, treatment of brain disorders is restricted by the blood-brain barrier (BBB), which is a highly selective membrane that protects the brain from exogenous substances. This study was undertaken to develop a novel strategy to overcome the BBB and improve the efficiency of drug delivery to the brain by mechanical load increase using hypergravity. Human cerebral microvascular endothelial cells were exposed three times to 20 min hypergravity (10g), with a 20-min rest period between each exposure. The applied hypergravity reversibly decreased the cellular metabolic activity and increased the permeation rate of fluorescein sodium salt, fluorescein isothiocyanate-labeled dextran (FD-4), and fluorescein-labeled jacalin. Following the exposure to hypergravity, we also observed structural changes of the cytoskeleton and tight junctions, and an alteration in the expression levels of related genes. These results indicate that increased mechanical load due to the applied hypergravity affects the cytoskeletal arrangement and tight junctions, thereby weakening the cell barrier function and enhancing the permeability of the paracellular pathway. Thus, the mechanical load increase by hypergravity has the potential of being used as a novel strategy to overcome the BBB for brain drug delivery.

Objectives/hypothesis This study was conducted to provide a vibrational culture model to investigate the effects of mechanical environments on cellular functions, and elucidate physiological characteristics of two different types of cells in vocal folds under static and vibrational conditions. Study design In vitro study of human vocal fold fibroblasts (hVFFs) and human macula flava stellate cells (hMF-SCs). Methods hVFFs and hMF-SCs were exposed to a 2-second-on/2-second-off, 205 Hz vibration regime for 4 hours by using a vibrational culture model. We compared cell morphology, cell viability, and gene expression in extracellular matrix-related components, growth factors, and differentiation markers under static and vibratory conditions. Results hVFFs and hMF-SCs differed in their morphologies and gene expression levels under static condition. The applied vibration did not induce changes in morphology and viability of either cell type. However, gene expression levels changed in both cell types in response to vibration; in particular, hMF-SCs exhibited a more sensitive response to vibration than that shown by hVFFs. Conclusions The vibrational culture model developed in this study enabled us to investigate the effects of the applied vibration on two types of vocal fold resident cells. As a result, we could demonstrate that hVFFs and hMF-SCs exhibited distinctively different characteristics under vibrational conditions. Level of evidence NA. Laryngoscope, 128:E258-E264, 2018.

CdSe quantum dots (QDs) with a uniform size distribution were synthesized using a droplet-based microfluidic reactor. The droplet-based microfluidic reactor enabled continuous production of CdSe QDs at a temperature of less than 250 °C in an extremely shorter reaction time (less than 30 s) when compared with the batch reactor. The photoluminescence (PL) and ultraviolet (UV) absorption spectra of the CdSe QDs were recorded at different reaction times and the size and optical properties of the QDs were discussed. The structure morphology and elemental composition of the CdSe QDs were determined using a transmission electron microscopy (TEM) and electrondispersive spectroscopy (EDS). The size of CdSe QDs prepared using the microfluidic reactor was estimated to be from 1.6 to 2.6 nm with an average size of 2.2 nm. This droplet-based microfluidic reactor has the potential to be automated system continuous synthesis of CdSe QDs.

Nasal route drug administration for local and systemic delivery of many therapeutics has received attention because the nasal cavity is highly vascularized and provides a large surface area for drug absorption. However, nasal mucosa exhibits limited permeability to polar molecules. In this study, we developed a novel method for improving absorption efficiency of polar drugs by applying hypergravity. RPMI 2650 cells and primary human nasal epithelial cells were exposed three times to a 20 min hypergravitational condition (10 × g) with a 20 min rest period after each exposure. The applied hypergravity induced a decrease in transepithelial electrical resistance without significant loss of cellular metabolic activity, and cellular permeability of fluorescein sodium salt (MW 376 Da; NaFI) and FITC-labeled dextran (average MW 4,000 Da; FD-4) increased by 19% and 16%, respectively. Immunostaining and RT-qPCR results demonstrated that hypergravity conditions affected cytoskeletal structures and tight junctions, leading to weakening of the cell barrier function and increasing the cellular permeability of polar molecules. Our results indicate that hypergravity could be used as a new strategy for enhancing the efficiency of drug absorption via the nasal route.

The inflammatory response is an indispensable bodily reaction, but excessive inflammation is known to result in diseases such as atopic disease, bronchitis, rheumatoid arthritis, and inflammatory bowel disease. Ceramide is the basic structure of sphingolipids and ceramides have been industrially used in functional cosmetics as anti-aging agents, as well as for moisturizing skin and calming skin irritation. It also has been recently used in medicinal fields as an anti-inflammatory as well as for atopic and skin wound healing, and for skin barrier restoration. In this study, we used genetically modified Saccharomyces cerevisiae to produce ceramides. Ceramide mixture was produced by gene manipulation that amplifies the original yeast gene. To investigate their anti-inflammatory effects, nitric oxide (NO) concentrations in cell culture supernatant were measured by using the Griess reaction and the expression levels of pro-inflammatory markers, cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α), were determined by using qRT-PCR. When cells were treated with the ceramide mixture, cell viability was not reduced, but NO production was inhibited. In addition, expressions of COX-2 and TNF-α were inhibited. Based on these results, we conclude that ceramide biosynthesized from recombinant yeast can effectively reduce the expression of inflammatory enzymes and cytokines. We expected that ceramides biosynthesized in genetically modified yeast is a novel preventive or therapeutic agent for inflammatory diseases without the risk of foreign gene introduction.

The improvement in the production of recombinant proteins has been linked in a number of small molecules such as carboxylic acids to the inhibition of histone deacetylase, leading to increased transcription of genes. However, carboxylic acids such as pentanoic acid and butanoic acid have been shown to promote an apoptotic response in Chinese hamster ovary (CHO) cell culture. Supplementation of cultures with antioxidants has shown the ability to reduce the apoptotic response of carboxylic acid supplementation, leading to increased therapeutic protein production. In this study, we showed that pentanoic acid reduced the number of cells entering early apoptosis relative to butanoic acid by 15.4%. Additionally, supplementation of butanoic acid- and pentanoic acid-treated cultures with N-acetyl cysteine (NAC) reduced the population of cells entering early apoptosis by 5.3 and 10.0%, respectively, while increasing productivity by 19.5% in the presence of pentanoic acid and NAC. Conversely, a decrease of 5.7% in production was observed in response to combined butanoic acid and N-acetyl cysteine treatment. The results presented herein provide evidence that a culture supplementation method is critical for optimization of biopharmaceutical manufacturing processes.

In the present study, poly(amidoamine)/5-fluorouracil (PAMAM/5-FU) was prepared and used as a conjugate system for delivering drugs to target E6 and E7 oncoproteins, which are predominant in cervical cancers. Specifically, molecular docking analysis was used to investigate the interaction between the PAMAM/5-FU and E6/E7 oncoproteins, which showed that the PAMAM/5-FU conjugate had a higher affinity for the oncoprotein than for 5-FU. Different generations of PAMAM dendrimers (0.5G, 1.0G, 1.5G, 2.0G, and 2.5G) were synthesized, characterized and tested as drug carriers for 5-FU. The PAMAM and PAMAM/5-FU drug conjugate showed less toxicity over COS-7 and HeLa cell lines. Laser confocal imaging and western blotting for tumor suppressor proteins pRb and p53 were used to confirm the interaction of PAMAM/5-FU with E6/E7 oncoproteins. Hematological analysis of PAMAM/5-FU using BALB/c female mice with cervical cancer confirmed the less toxic nature of this material. Based on these results, the developed PAMAM/5-FU conjugate is a potential candidate for the treatment of cervical cancer.

Adenylate cyclase is a key intracellular enzyme involved in energy imbalance leading to tumor hypoxia and cytotoxicity. In this study, adenylate cyclase activities in isolated hepatocytes and Kupffer cells were compared in the presence of several metabolic stimulators. In cultured hepatocyte cells, adenylate cyclase was stimulated by guanylyl imidotriphosphate (GITP), guanosine triphosphate (GTP), progesterone and nitroimidazole embedded nanoparticle (NNP) effectors, while prostaglandin E2 and F2α were used as effectors in cultured Kupffer cells. The results showed that NNPs decreased adenylate cyclase specific activity in a dose-dependent manner after preincubation of hepatocytes with NNPs. The NNPs stimulated adenylate cyclase activities in hepatocytes were evaluated based on measurement of cyclic adenosine monophosphate (cAMP). The stimulatory effects of NNPs on adenylate cyclase were independent of the presence of GTP and may have been due to a direct effect on the catalytic subunit of adenylate cyclase. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the NNPs. In conclusion, NNPs exerted direct effects on the catalytic subunit of the adenylate cyclase system, and adenylate cyclase was hormone sensitive in liver cells.

The vocal folds (VFs) are connective tissues with complex matrix structures that provide the required mechanical properties for voice generation. VF injury leads to changes in tissue structure and properties, resulting in reduced voice quality. However, injury-induced biochemical changes and repair in scarred VF tissues have not been well characterized to date. To treat scarred VFs, it is essential to understand how physiological characteristics of VFs tissue change in response to external perturbation. In this study, we designed a simple vibrational culture model to mimic vibratory microenvironments observed in vivo. This model consists of a flexible culture plate, three linear actuators, a stereo splitter, and a function generator. Human vocal fold fibroblast (hVFF) monolayers were established on the flexible membrane, to which normal phonatory vibrations were delivered from linear actuators and a function generator. The hVFF monolayers were exposed to the vibrational stresses at a frequency of 205 Hz for 2, 6, and 10 h with maximum displacement of 47.1 μm, followed by a 6 h rest. We then observed the changes in cell morphology, cell viability, and gene expression related to extracellular matrix components. In our dynamic culture device mimicking normal phonatory frequencies, cell proliferation increased and expression of hyaluronic acid synthase 2 was downregulated in response to vibrational stresses. The results presented herein will be useful for evaluating cellular responses following VF injuries in the presence or absence of vibrational stresses.

As the outermost organ, the skin can be damaged following injuries such as wounds and bacterial or viral infections, and such damage should be rapidly restored to defend the body against physical, chemical, and microbial assaults. However, the wound healing process can be delayed or prolonged by health conditions, including diabetes mellitus, venous stasis disease, ischemia, and even stress. In this study, we developed a vibrational cell culture model and investigated the effects of mechanical vibrations on human keratinocytes. The HaCaT cells were exposed to vibrations at a frequency of 45 Hz with accelerations of 0.8g for 2 h per day. The applied mechanical vibration did not affect cell viability or cell proliferation. Cell migratory activity did increase following exposure to vibration, but the change was not statistically significant. The results of immunostaining (F-actin), western blot (ERK1/2), and RT-qPCR (FGF-2, PDGF-B, HB-EGF, TGF-β1, EGFR, and KGFR) analyses demonstrated that the applied vibration resulted in rearrangement of the cytoskeleton, leading to activation of ERK1/2, one of the MAPK signaling pathways, and upregulation of the gene expression levels of HB-EGF and EGFR. The results suggest that mechanical vibration may have wound healing potential and could be used as a mechanical energy-based treatment for enhancing wound healing efficiency.

The success of cell-based therapies for treatment of liver disease hinges on the phenotypic stability of isolated hepatocytes; however, primary hepatocytes rapidly lose many liver-specific functions following enzymatic isolation from the liver. Previous studies have demonstrated that liver-specific functions can be stabilized by cocultivation with non-parenchymal cells. Although the precise mechanisms underlying these effects have not yet been elucidated, it is believed that gap junctional intercellular communication plays an important role in cell-cell communication. In this study, indirect immunofluorescence was used to identify connexin 32 and 26 as homotypic gap junction proteins that were present between hepatocytes during co-culture with 3T3 fibroblasts, but not in pure hepatocyte cultures. Additionally, dye transfer of Lucifer yellow revealed that these gap junctions were functionally coupled. Although previous studies reported abundant connexin 43 expression in 3T3 fibroblasts and functional coupling by dye transfer, we did not observe heterotypic gap junction (connexin 43) expression or its functional coupling by either immunofluorescence or dye transfer. Incubation with a gap junction inhibitor, 18β-glycyrrhetinic acid, in hepatocyte co-culture diminished the level of liverspecific markers such as albumin production and P450 activity relative to control co-cultures. Overall, these findings suggest that homotypic gap junctions are present in hepatocytes co-cultured with fibroblasts and might play a functional role in homotypic hepatocyte communication. In conclusion, homotypic gap junctional communication may be important to phenotypic stability of isolated hepatocytes in co-culture and crucial to the design of tissue-engineered therapies for treatment of liver failure.

Most studies on the adverse effects of air pollution have focused on respiratory and cardiovascular diseases, and there are relatively few studies on eye diseases following exposure of ambient particulate matter (PM). Epidemiological and clinical researches correlating the eye and PMs have recently received attention. PMs are complex mixture of particles that vary in chemical composition and size. This study investigated the influence of collected road dust on cell viability, inflammatory responses, and oxidative stress in human corneal epithelial cells. The collected road dust was classified with respect to aerodynamic diameter and solubility. Exposure concentration was calculated based on the particle deposition rate. We observed a dose-dependent decrease in cell viability at total PM2.5 and PM10. The pellet fractions of total PM2.5 and PM10 mainly contributed to the mitochondrial activity. Although both total PM2.5 and PM10 did not change the membrane integrity, the supernatant fractions significantly affected cell membrane integrity. Both total and fractions induced nitric oxide production and interleukin 8 expression. In addition, total PM2.5 and PM10 increased the oxidative stress; the pellet fractions of total PM2.5 and PM10 also induced higher oxidative stress. However, there was no significant difference between the cellular responses of total PM2.5 and PM10. We observed that the effects of collected road dust on cellular responses were strongly dependent on their concentration and solubility.

We investigated whether the therapeutic effects of dexamethasone for allergic asthma and rhinitis were enhanced in mice when exposed to hypergravity. Forty mice were divided into 5 groups (n = 8/group): Control group received saline intraperitoneally (i.p.) and intranasally (i.n.); Asthma group received i.p./i.n. ovalbumin (OVA) for inducing allergic asthma/rhinitis; Dexa group received i.n. dexamethasone (0.75 mg/kg) 30 minutes before each OVA challenge; Hypergravity group was subjected to allergic asthma/rhinitis as well as exposed to 5 G hypergravity for 30 days; Finally in Dexa/Hypergravity group, hypergravity and dexamethasone were used simultaneously during induction of allergic asthma/rhinitis. Dexa group and Hypergravity group showed a significant decrease in serum total IgE levels compared to the Asthma group (p

Inhibition of mammalian target of rapamycin (m- TOR) is a potential method for cancer treatment. Effects of rapamycin (RAP) on the reversion of malignant breast epithelial cells were investigated on three-dimensional (3D) basement membrane extract (BME) cultures. Through continuous exposure to 20 nM of RAP, cell colony size was significantly reduced in 3D BME cultures of malignant breast epithelial cells, while normal cell colony size appeared unaffected. In unfixed 3D BME cultures of normal and RAP-treated malignant breast epithelial cells, the presence of luminal cell death was confirmed by ethidium bromide and propidium iodide labeling. Increased structural organization was observed by im- munofluorescence staining of F-actin and β-catenin in RAP-treated malignant breast epithelial cells. In monolayer cultures of normal and malignant breast epithelial cells, continuous exposure to 20 nM of RAP increased caspase 3/7 activity and decreased proliferation. Reverse transcriptase polymerase ch- ain reaction (RT-PCR) array analysis indicated a fold increase in the expression of a number of proteins related to polarity, cell-cell adhesion, and cell-matrix adhesion in the presence of RAP. Our data showed that phenotypic reversion of malignancy can be ach- ieved through RAP exposure on 3D BME cultures. This 3D BME culture system will provide correct microenvironments for observing the effects of other mTOR inhibitors on phenotypic reversion of malignant breast epithelial cells.

The anti-inflammatory effects of the new ster-oidal antedrug, 21-acetyloxy-9α-fluoro-11β-hy-droxyl-3, 20-dioxo-1, 4-pregnadieno-[16α, 17α-d] isoxazoline (FP-ISO-21AC), on nitric oxide (NO) and interleukin 8 (IL-8) production, were inves-tigated together with its parent steroid predni-solone (PRED). PRED is one of the anti-in-flammatory steroids but has systemic side ef-fects which limit the use of it. PRED was modi-fied with ‘antedrug concept’ to create safer drugs that attack problems such as inflamma-tion, then quickly become inactive before they can cause systemic side effect. We had a test about the effect of the modified anti-inflamma-tory steroidal antedrug on anti-inflammatory activity. The present study evaluated their ability to inhibit cytokine-induced NO and IL-8 produc-tion in human alveolar epithelial cells. We also investigated their ability to enhance the expres-sion of inhibitory cytokine receptor, interleukin 22 receptor (IL-22R) in human alveolar epithelial cells. Our results showed that FP-ISO-21AC sh- owed higher ability to inhibit the cytokine - in-duced production of NO than PRED. Exogenous IL-22 was added to the media of both human alveolar epithelial cells (A549) and human lung fibroblast (HLF-1). In the presence of the ex-ogenous inhibitory cytokine IL-22, further re-duction of NO production was observed in A549 cells, which express IL-22R, but not in HLF1, which does not express IL-22R. These data suggested that the steroidal antedrugs en-hanced the expression of IL-22R. FP-ISO- 21AC showed higher potency than PRED to restore the expression of IL-22R. FP-ISO-21AC further reduced NO production to 27% and PRED further reduced NO production to 39%. In con-clusion, a synthesized steroidal antedrug FP- ISO-21AC showed higher anti-inflammatory ef-fects than PRED by inhibiting the expression of pro-inflammatory mediator NO and stimulating the expression of IL-22R.

Recent discoveries of various forms of carbon nanostructure have stimulated research on their applications and hold promise for applications in medicine and other related engineering areas. Although carbon nanotubes (CNTs) are already being produced on a massive scale, few studies have been performed which test the potential harmful effects of this new technology. The authors used a three-dimensional in vitro model of the human airway using a coculture of normal human bronchial epithelial cells and normal human fibroblasts for the health risk assessment of CNTs on the human respiratory systems. The authors used aqueous single-walled carbon nanotube (SWCNT) solution. The average length and diameter of nanotube ropes were about 500 nm and less than 10 nm, respectively. The authors measured the production of nitric oxide (NO) as an inflammatory marker and mitochondrial activity using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as a cytotoxic response of the cell layers following exposure of different concentration of aqueous SWCNT solution. The results indicated that NO production was dramatically increased and cell viability was decreased following exposure of different concentrations of SWCNTs. Transepithelial electrical resistance (TER) across the coculture layers was measured to observe the changes in airway physiological function following exposure of different concentrations of SWCNTs. TER value was dramatically decreased following exposure of 20% SWCNT (8 μg/ml). In this study, the authors presented viable alternatives to in vivo tests to evaluate the toxicity of engineered SWCNTs. Cytotoxic/inflammatory responses and barrier function of the human lung layers following exposure of SWCNTs were observed using in vitro coculture system of airway.

Alzheimer’s disease (AD) is considered a slow neuronal dysfunction process through hypoxia, ischemia and leads to apoptosis mediated senile plaques and neurofibrillary tangles (NFTs). Due to non-invasive approach of plaque characterization, computational techniques based on Brownian dynamics simulation are unique to speculate the electrostatic and kinetic properties of Acetylcho-linesterase (AChE). Typically the MRI spectros-copy high choline peak and enzyme specific to Alzheimer’s Disease (specificity constant (kcat/Km) of AChE) appeared associated with apoptosis and hypoxia. A simple display between synergy of cytokines, apoptosis, elevated AChE and choline is postulated as initial events. The events may be distributed heterogeneously within the senile plaques and neurofibrillary tangles (NFTs) of Alzheimer’s Disease (AD). The role of decreased brain AChE and synergy was associated with specific Magnetic Resonance Spectroscopic (MRS) pattern profiles in AD. These findings suggest that that the altered AChE and early apoptosis events in AD may be associated with specific MR spectral peak patterns. This study opens the possibility of reduced AChE levels causing high choline and reduced N-acetyl ace-tate (NAA) neurotransmitter by MRS after initial apoptosis and/or inflammation to make amyloid plaques in the cerebral tissue of Alzheimer’s disease (AD) patients. These results can be useful in clinical trials on AD lesions.

Nanotechnology involves working at the atomic, molecular, and macromolecular levels including imaging. Recently, four areas have emerged in cardiovascular imaging: 1. Targeted therapeutics to deliver cardioprotective drugs at the target sites they are needed; 2. Myocardial tissue engineering to replace the defective valves, damaged heart muscle, clogged blood vessels and myocardium; 3. Molecular imaging using “smart” imaging agents in targeted therapeutics and imaging; 4. Biosensors and myocardial diagnostics. Several approaches to nanoparticles i.e. dendrimers, liposomes, polymer delivery molecules, cantilevers, nanoscaffolds, nanofibers are potential candidates in cardiac visualization. The extracellular matrix plays a significant role by chemokines, cytokines and growth factors. The limitations of these emerging techniques and a new possibility of MRI visualization of mouse cardiac atheroma by superparamagnetic iron-oxide gadolinium-apoferritin (SPIOA) and myoglobin (SPIOM), for targeted functional and...

Nitric oxide (NO) biosensors are novel tools for real-time bioimaging of tissue oxygen changes and physiological monitoring of tissue vasculature. Nitric oxide behavior further enhances its role in mapping signal transduction at the molecular level. Spectrometric electron paramagnetic resonance (EPR) and fluorometric imaging are well known techniques with the potential forin vivobioimaging of NO. In tissues, NO is a specific target of nitrosyl compounds for chemical reaction, which provides a unique opportunity for application of newly identified NO biosensors. However, the accuracy and sensitivity of NO biosensors still need to be improved. Another potential magnetic resonance technique based on short term NO effects on proton relaxation enhancement is magnetic resonance imaging (MRI), and some NO biosensors may be used as potent imaging contrast agents for measurement of tumor size by MRI combined with fluorescent imaging. The present review provides supporting information regarding the possible use of nitrosyl compounds as NO biosensors in MRI and fluorescent bioimaging showing their measurement limitations and quantitative accuracy. These new approaches open a perspective regarding bioimaging of NO and thein vivoelucidation of NO effects by magnetic resonance techniques.

Recombinant Escherichia coli was cultured up to OD600 90 with glycerol fed by the DO-STAT method to produce recombinant beta-lactamase. Optimum feeding and induction conditions were determined. When glycerol was maintained at a high concentration (40-50 g/L) after isopropyl thiogalactoside (IPTG) induction at OD600 75, a 2-fold increase in the amount of soluble beta-lactamase was obtained. Similarly, a 3-fold increase was obtained by the addition of NaCl (0.4 M) at low glycerol concentration (10 g/L).

Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different.

Diesel particulate matter (DPM) possesses the potential to induce acute and chronic health issues upon occupational and daily exposure. Many recent studies have focused on understanding molecular mechanisms to depict DPM's side effects inside the lung using static in vitro cell culture models. These studies have provided abundant fundamental information on DPM's adverse effects on cellular responses, but these systems were limited by the absence of dynamic nature to access relevant cellular responses and functionality. We hypothesized that the exposure of DPM under dynamic environment may affect the levels of cellular inflammation and reactive oxygen species, which may be different from those under static environments. In this study, we used the dynamic cell growth condition to mimic mechanically dynamic environment similar to the normal breathing in vivo. We also used high (20, 10, and 5 ppm) and low (3, 1, 0.1, and 0.01 ppm) ranges of DPM exposure to mimic different levels of exposure, respectively. Following 24-, 48-, and 72-h exposure of DPM, Interleukin-8 (IL-8), C-reactive protein (CRP), reactive oxygen species (ROS), and total amount of protein were analyzed. Our results demonstrated the distinct differences in the profiles of inflammatory mediators (IL-8, CRP, and ROS) between the static and dynamic cell growth conditions.

Effects ofin-vitro protein stabilizers, such as glycerol, fructose and NaCl, were investigated on the overproduction of the secreted recombinant protein, β-lactamas, inE. coli. With the addition of glycerol, a five-fold increase in the amount of soluble β-lactamase was obtained under optimal conditions. The extent of excretion was dependent on the glycerol concentration. Fructose showed similar effects. The effect of NaCl was dependent on the carbon source used.

Background Silver has long been known to have antimicrobial activity. To incorporate this property into multiple applications, a silver-impregnated cellulose (SIC) with low cytotoxicity to human cells was developed. SIC differs from other silver treatment methods in that the leaching of silver particles is non-existent and the release of ionic silver is highly controlled. Results Candida albicans, Micrococcus luteu, Pseudomonas putida, and Escherichia coli were used for antimicrobial testing. No microbial cells were able to grow in the presence of SIC at concentrations above 0.0035 Ag w/v %. Even at a concentration of 0.00035 Ag w/v %, P. putida and M. luteu failed to grow, and C. albicans and E. coli exhibited diminished growth. To determine the cytotoxic effect of silver on human cells, five different concentrations of SIC were tested on human fibroblasts. In SIC concentrations of 0.035 Ag w/v % and below, no cytotoxicity was observed. Conclusion The optimal concentration of SIC for a broad range of anti-microbial activity and low or negligible cytotoxicity was 0.0035 Ag w/v %. Although the highly controlled releasing characteristics of SIC would prove a substantial improvement over current technologies, further investigation for genotoxicity and other biocompatibility test will be required.

Ventilator-induced lung overdistension has been a growing concern in the management of mechanically ventilated patients. Mechanical ventilation triggers or enhances the net inflammatory and tissue remodeling activities. Although it has been shown that proinflammatory and tissue remodeling factors play important roles during airway remodeling, the interplay between them is not well understood. Thus, our objective was to study and characterize the molecular mechanism of cyclic equibiaxial deformation-induced airway inflammation and remodeling either in the presence or absence of a pre-existing inflammatory condition. This study was done using an in vitro dynamic model, which can simulate different mechanical ventilative conditions. Type II alveolar epithelial cell (A549) monolayers were exposed to the different levels of mechanical ventilative conditions using the Flexcell® Tension Plus™ 4000T system, which generated the different levels of cyclic equibiaxial deformation (5, 10, 15, and 20%) at 0.2 Hz deformation frequency. The production of nitric oxide (NO), the expression of metalloprotease-2 (MMP-2)/tissue inhibitor metalloprotease-2 (TIMP-2), and the activation of MMP-2 were measured under the different levels of cyclic equibiaxial deformation either in the presence or absence of TNF-α. Our study indicated that cyclic equibiaxial deformation-induced production of NO and MMP-2/TIMP-2. Higher levels of cyclic equibiaxial deformation increased the expression of the active form of MMP-2. In particular, in the presence of TNF-α, the more active form of MMP-2 was detected during both cyclic equibiaxial deformation and remodeling periods.

Nitroimidazole is an antibiotic and radiosensitizer chemical with great potentials in imaging. The hepato-cytoxicity evaluation of nitroimidazole and possible energy status changes in liver cells due to its anti-inflammatory characteristics are crucial in tumor imaging and chemotherapy. Adenylate cyclase is key enzyme to cause energy imbalance leading to cytotoxicity. To evaluate energy status in hepatocytes and Kupffer cells, adenylate cyclase activities in isolated liver cells were compared in presence of stimulators. To evaluate the effect of effectors on adenylate cyclase in cultured hepatocyte cells, adenylate cyclase enzyme was stimulated by different GITP, GTP, progesterone and nitroimidazole effectors. In cultured Kupffer cells, prostaglandin E~2~ and F~2[alpha]~ were used as effectors. The results showed that nitroimidazole decreased adenylate cyclase specific activity in dose-dependent manner after pre-incubation of hepatocytes with the nitroimidazole in medium. Nitroimidazole stimulated adenylate cyclase activities in hepatocytes were mediated by cAMP and determined by cAMP mesurement. The stimulatory effect of nitroimidazole on adenylate cyclase was independent of the GTP presence in the assay system perhaps due to a direct effect on the catalytic subunit of adenylate cyclase enzyme. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the nitroimidazole. In conclusion, nitroimidazole exhibits direct effect on the catalytic subunit of the adenylate cyclase system. The adenylate cyclase was hormone sensitive in liver cells.

Nitric oxide (NO) derived from inducible NO synthase (iNOS) at sites of inflammation is closely related to host defense against infection and airway inflammation. Cytokines are known to stimulate NO production in human alveolar epithelial cells in a synergistic (nonlinear or nonadditive) manner. The mechanism of this synergy is not known. We measured the activation of the transcription factor NF-kappaB, the iNOS protein, and NO production in A549 monolayers (human alveolar epithelial cell line) in response to different combinations of IL-1beta, INF-gamma, and TNF-alpha (100 ng/ml), and the cofactors FMN, FAD, and BH4. We found that both IL-1beta and TNF-alpha could independently activate cytosolic NF-kappaB, direct its translocation into the nucleus, and induce iNOS monomer synthesis. In addition, different combinations of cytokines produced synergistic amounts of iNOS monomers. Exogenous BH4 (0.1 microM) had no impact on NO production induced by cytokine combinations that included IL-1beta, but significantly enhanced NO production in the presence of INF-gamma and TNF-alpha, and allowed TNF-alpha independently to produce NO. We conclude that there are at least three mechanisms of synergistic cytokine-induced NO production: (1) the biosynthesis of iNOS monomer due to nonlinear interactions by transcription factors, (2) synergistic cytosolic activation of NF-kappaB, and (3) parallel biosynthesis of BH4 in the presence of cytokine combinations that include IL-1beta.

Nitric oxide (NO) is an important mediator molecule in regulating normal airway function, as well as in the pathophysiology of inflammatory airway diseases. In addition, cytokines are potent messenger molecules at sites of inflammation. The specific relationship among IL-1beta, TNF-alpha, and IFN-gamma on iNOS induction and NO synthesis in human alveolar epithelial cells has not been determined. In addition, rigorous methods to determine potential synergistic action between the cytokines have not been employed. We exposed monolayer cultures of A549 cells to a factorial combination of three cytokines (IL-1beta, TNF-alpha, and IFN-gamma) and three concentrations (0, 5, and 100 ng/mL). TNF-alpha alone does not induce NO production directly; however, it does have a stimulatory effect on IL-1beta-induced NO production. IL-1beta and INF-gamma both induce NO production alone, yet at different concentration thresholds, and act synergistically when present together. In the presence of all three cytokines, the net effect of NO production exceeds the predicted additive effect of each individual cytokine and the two-way interactions. Several plausible mechanisms of synergy among IL-1beta, TNF-alpha, and IFN-gamma in NO production from human alveolar epithelial cells (A549) are proposed. In order to verify the proposed mechanisms of synergy, future experimental and theoretical studies must address several molecular steps through which the iNOS gene is expressed and regulated, as well as the expression and regulation of enzyme cofactors and substrates.

Background Rapid increase in multi-walled carbon nanotube (MWCNT) production for their industrial and biomedical applications has led to concerns over the effects of MWCNTs on human health and the environment. Both animal and in vitro studies have provided important findings about MWCNT-induced effects on the lung cells or tissues. In vitro studies have provided a considerable amount of fundamental information on MWCNT-induced effects on the specific lung cells. However, the cell culture systems used in those studies were limited by the absence of dynamic nature of lung tissues. We hypothesized that MWCNT-induced cellular responses such as proliferation, inflammation, and oxidative stress under dynamic cell growth environment may differ from those under static cell growth environment. Results In this study, we used a dynamic cell growth condition to mimic mechanically dynamic environment of the lung and characterized interleukin 8 (IL-8), reactive oxygen species (ROS), glutathione (GSH), and cell proliferation for three days following exposure of MWCNTs at different concentrations (5, 10, and 20 μg/ml) to A549 cell monolayer under both static and dynamic cell growth conditions. Our results demonstrated the distinct differences in the levels of inflammatory response and oxidative stress between static and dynamic cell growth conditions. Conclusions In conclusion, the dynamic cell growth system used in this study provided important changes in cellular responses that were not found in the static cell growth system and were similar to animal studies. The dynamic cell growth system can be considered as a viable alternative to in vivo test system in combination with existing in vitro static cell growth systems to evaluate the effect of MWCNTs on cellular responses in the respiratory system.