Synthesis, characterization and in vitro biocompatibility assessment of a novel tripeptide hydrogelator, as a promising scaffold for tissue engineering applications.

PubMed

Pospišil, Tihomir; Ferhatović Hamzić, Lejla; Brkić Ahmed, Lada; Lovrić, Marija; Gajović, Srećko; Frkanec, Leo

2016-10-20

We have synthesized and characterized a self-assembling tripeptide hydrogelator Ac-l-Phe-l-Phe-l-Ala-NH2. A series of experiments showed that the hydrogel material could serve as a stabile and biocompatible physical support as it improves the survival of HEK293T cells in vitro, thus being a promising biomaterial for use in tissue engineering applications.

[The in vitro action of plants on Vibrio cholerae].

PubMed

Guevara, J M; Chumpitaz, J; Valencia, E

1994-01-01

Natural products of several plants, according to the geographic location, are used by Peruvian people in the popular treatment of diarrhea, with good success. When cholerae cases appeared in Peru, we were interested to know the "in vitro" effect against Vibrio cholerae 01, of these useful plants to treat diarrhea. The following plants were tested: Cichorium intybus, Althaea officinalis, Psorela glandulosa, Geranium maculatum, Punica granatum, Malus sativa, Cydonia oblonga, Chenopodium ambrosoides, Krameria triandria, Tea chinensis, Daucus carota, Persea gratissima, Psidium guayaba and Lippia dulcis. Decoction or infusion of the plants were used in the "in vitro" experiments. The following plants showed no "in vitro" effect against V. cholerae: Cichorium intybus, Althaea officinalis, Psorela glandulosa, Geranium maculatum, Chenopodium ambrosoides, Krameria triandria, Psidium guayaba, Lippia dulcis and Daucus carota. Decoction of Malus sativa and Cydenia oblonga showed bactericidal effect for their acidity and stone avocado (Persea gratissima) a late bactericidal effect. Tea infusión and the decoction of Punica granatum peel, showed the best bactericidal effect and we suggest to use them as to stop cholera spreading.

Calcification of in vitro developed hypertrophic cartilage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tacchetti, C.; Quarto, R.; Campanile, G.

1989-04-01

We have recently reported that dedifferentiated cells derived from stage 28-30 chick embryo tibiae, when transferred in suspension culture in the presence of ascorbic acid, develop in a tissue closely resembling hypertrophic cartilage. Ultrastructural examination of this in vitro formed cartilage showed numerous matrix vesicles associated with the extracellular matrix. In the present article we report that the in vitro developed hypertrophic cartilage undergoes calcification. We indicate a correlation between the levels of alkaline phosphatase activity and calcium deposition at different times of development. Following the transfer of cells into suspension culture and an initial lag phase, the level ofmore » alkaline phosphatase activity rapidly increased. In most experiments the maximum of activity was reached after 5 days of culture. When alkaline phosphatase activity and /sup 45/Ca deposition were measured in the same experiment, we observed that the increase in alkaline phosphatase preceded the deposition of nonwashable calcium deposits in the cartilage.« less

Montreal electronic artificial urinary sphincters: Our futuristic alternatives to the AMS800™.

PubMed

Biardeau, Xavier; Hached, Sami; Loutochin, Oleg; Campeau, Lysanne; Sawan, Mohamad; Corcos, Jacques

2017-10-01

We aimed to present three novel remotely controlled hydromechanical artificial urinary sphincters (AUSs) and report their in-vitro and ex-vivo results. We successively developed three distinct hydromechanical AUSs on the basis of the existing AMS800 ™ device by incorporating an electronic pump. No changes were made to the cuff and balloon. The AUS#1 was designed as an electromagnetically controlled device. The AUS#2 and AUS#3 were conceived as Bluetooth 2.1 remotely controlled and Bluetooth 4.0 remotely-controlled, adaptive devices, respectively. In-vitro experiments profiled occlusive cuff pressure (OCP) during a complete device cycle, with different predetermined OCP. Ex-vivo experiments were performed on a fresh pig bladder with 4 cm cuff placed around the urethra. Leak point pressure with different predetermined OCP values was successively measured during cystometry via a catheter at the bladder dome. Our in-vitro and ex-vivo experiments demonstrated that these three novel AUSs provided stable and predetermined OCP - within the physiological range - and completely deflated the cuff, when required, in a limited time compatible with physiological voiding cycles. Our three novel, remotely controlled AUSs showed promising results that should be confirmed by in-vivo experiments focusing on efficacy and safety.

Fiber optic backscatter spectroscopic sensor to monitor enamel demineralization and remineralization in vitro

PubMed Central

Kishen, Anil; Shrestha, Annie; Rafique, Adeela

2008-01-01

In this study, a Fiber Optic Backscatter Spectroscopic Sensor (FOBSS) is used to monitor demineralization and remineralization induced changes in the enamel. A bifurcated fiber optic backscatter probe connected to a visible light source and a high resolution spectrophotometer was used to acquire the backscatter light spectrum from the tooth surface. The experiments were conducted in two parts. In Part 1, experiments were carried out using fiber optic backscatter spectroscopy on (1) sound enamel and dentine sections and (2) sound tooth specimens subjected to demineralization and remineralization. In Part 2, polarization microscopy was conducted to examine the depth of demineralization in tooth specimens. The enamel and dentine specimens from the Part-1 experiments showed distinct backscatter spectra. The spectrum obtained from the enamel-dentine combination and the spectrum generated from the average of the enamel and dentine spectral values were closely similar and showed characteristics of dentine. The experiments in Part 2 showed that demineralization and remineralization processes induced a linear decrease and linear increase in the backscatter light intensity respectively. A negative correlation between the decrease in the backscatter light intensity during demineralization and the depth of demineralization determined using the polarization microscopy was calculated to be p = -0.994. This in vitro experiment highlights the potential benefit of using FOBSS to detect demineralization and remineralization of enamel. PMID:20142887

[Study on solid dispersion of precipitated calcium carbonate-based oleanolic acid].

PubMed

Yan, Hong-mei; Zhang, Zhen-hai; Jia, Xiao-bin; Jiang, Yan-rong; Sun, E

2015-05-01

Oleanolic acid-precipitated calcium carbonate solid dispersion was prepared by using solvent evaporation method. The microscopic structure and physicochemical properties of solid dispersion were analyzed using differential scanning calorimetry and scanning electron microscopy (SEM). And its in vitro release also was investigated. The properties of the precipitated calcium carbonate was studied which was as a carrier of oleanolic acid solid dispersion. Differential scanning calorimetry analysis suggested that oleanolic acid may be present in solid dispersion as amorphous substance. The in vitro release determination results of oleanolic acid-precipitated calcium carbonate (1: 5) solid dispersion showed accumulated dissolution rate of.oleanolic acid was up to 90% at 45 min. Accelerating experiment showed that content and in vitro dissolution of oleanolic acid solid dispersion did not change after storing over 6 months. The results indicated that in vitro dissolution of oleanolic acid was improved greatly by the solid dispersion with precipitated calcium carbonate as a carrier. The solid dispersion is a stabilizing system which has actual applied value.

In vitro and in vivo biological activities of anthocyanins from Nitraria tangutorun Bobr. fruits.

PubMed

Ma, Tao; Hu, Na; Ding, Chenxi; Zhang, Qiulong; Li, Wencong; Suo, Yourui; Wang, Honglun; Bai, Bo; Ding, Chenxu

2016-03-01

Anthocyanins are the main compounds in Nitraria tangutorun Bobr. The enrichment and purification of anthocyanins on macroporous resins were investigated. Regarding anthocyanin purification, static adsorption and desorption were studied. The optimal experimental conditions were the following: resin type: X-5; static adsorption time: 6h; desorption solution: ethanol-water-HCl (80:19:1, V/V/V; pH 1); desorption time: 40min. Furthermore, the in vitro and in vivo biological activities of the anthocyanins were evaluated. The anthocyanins showed ideal scavenging effects on free radicals in vitro, especially on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl free radical (OH). In the animal experiment, blood lipid metabolism of hyperlipidemia rats was regulated by anthocyanin contents. The superoxide dismutase (SOD) activity and the total antioxidant capacity (TAC) of hyperlipidemia rats were also improved by anthocyanins. These results showed that anthocyanins from N. tangutorun Bobr. fruits had potential biological activities in vivo as well as in vitro. Copyright © 2015. Published by Elsevier Ltd.

Proton pump inhibitors while belonging to the same family of generic drugs show different anti-tumor effect.

PubMed

Lugini, Luana; Federici, Cristina; Borghi, Martina; Azzarito, Tommaso; Marino, Maria Lucia; Cesolini, Albino; Spugnini, Enrico Pierluigi; Fais, Stefano

2016-08-01

Tumor acidity represents a major cause of chemoresistance. Proton pump inhibitors (PPIs) can neutralize tumor acidity, sensitizing cancer cells to chemotherapy. To compare the anti-tumor efficacy of different PPIs in vitro and in vivo. In vitro experiments PPIs anti-tumor efficacy in terms of cell proliferation and cell death/apoptosis/necrosis evaluation were performed. In vivo PPIs efficacy experiments were carried out using melanoma xenograft model in SCID mice. Lansoprazole showed higher anti-tumor effect when compared to the other PPIs. The lansoprazole effect lasted even upon drug removal from the cell culture medium and it was independent from the lipophilicity of the PPIs formulation. These PPIs have shown different anti-tumoral efficacy, and the most effective at low dose was lansoprazole. The possibility to contrast tumor acidity by off-label using PPIs opens a new field of oncology investigation.

[Preparation of ibuprofen/EC-PVP sustained-release composite particles by supercritical CO2 anti-solvent technology].

PubMed

Cai, Jin-Yuan; Huang, De-Chun; Wang, Zhi-Xiang; Dang, Bei-Lei; Wang, Qiu-Ling; Su, Xin-Guang

2012-06-01

Ibuprofen/ethyl-cellulose (EC)-polyvinylpyrrolidone (PVP) sustained-release composite particles were prepared by using supercritical CO2 anti-solvent technology. With drug loading as the main evaluation index, orthogonal experimental design was used to optimize the preparation process of EC-PVP/ibuprofen composite particles. The experiments such as encapsulation efficiency, particle size distribution, electron microscope analysis, infrared spectrum (IR), differential scanning calorimetry (DSC) and in vitro dissolution were used to analyze the optimal process combination. The orthogonal experimental optimization process conditions were set as follows: crystallization temperature 40 degrees C, crystallization pressure 12 MPa, PVP concentration 4 mgmL(-1), and CO2 velocity 3.5 Lmin(-1). Under the optimal conditions, the drug loading and encapsulation efficiency of ibuprofen/EC-PVP composite particles were 12.14% and 52.21%, and the average particle size of the particles was 27.621 microm. IR and DSC analysis showed that PVP might complex with EC. The experiments of in vitro dissolution showed that ibuprofen/EC-PVP composite particles had good sustained-release effect. Experiment results showed that, ibuprofen/EC-PVP sustained-release composite particles can be prepared by supercritical CO2 anti-solvent technology.

Impact of ketorolac administration around ovarian stimulation on in vivo and in vitro fertilization and subsequent embryo development.

PubMed

Jee, Byung Chul; Youm, Hye Won; Lee, Jae Ho; Kim, Jee Hyun; Suh, Chang Suk; Kim, Seok Hyun

2013-05-01

We performed this study to investigate the effect of ketorolac (a non-steroidal anti-inflammatory drug) administration around ovarian stimulation on in vivo and in vitro fertilization process. Sixty-four female mice (ICR) were injected with ketorolac (0, 7.5, 15 and 30 µg/d) for 3 d starting from the day of eCG treatment. In experiment 1, 41 mice were triggered by hCG and then mated; two-cell embryos were obtained and in vitro development up to blastocyst was observed. In experiment 2, 23 mice were triggered by hCG and mature oocytes were collected; in vitro fertilization rate and subsequent embryo development up to blastocyst was recorded. In experiment 1, the blastocyst-forming rates per in vivo fertilized two-cell embryo showed an inverse relationship with a dosage of ketorolac (97.6%, 64.2%, 35.4% and 25.9%). In experiment 2, degenerated oocytes were frequently observed in a dose-dependent manner (4.3%, 22.9%, 22.4% and 75.0%). Lower fertilization rates were noted in all the three ketorolac-treating groups; blastocyst-forming rate was significantly lower in 30-µg-treating group when compared with the control group. Administration of ketorolac around ovarian stimulation significantly affects the development of in vivo fertilized embryo in a dose-dependent manner. High-dose ketorolac could result in a poor oocyte quality and decreased embryo developmental competence.

Concentration-Dependent Inhibitory Effect of Baicalin on the Plasma Protein Binding and Metabolism of Chlorzoxazone, a CYP2E1 Probe Substrate, in Rats In Vitro and In Vivo

PubMed Central

Gao, Na; Zou, Dan; Qiao, Hai-Ling

2013-01-01

Some of the components found in herbs may be inhibitors or inducers of cytochrome P450 enzymes, which may therefore result in undesired herb-drug interactions. As a component extracted from Radix Scutellariae, the direct effect of baicalin on cytochrome P450 has not been investigated sufficiently. In this study, we investigated concentration-dependent inhibitory effect of baicalin on the plasma protein binding and metabolism of chlorzoxazone (CZN), a model CYP2E1 probe substrate, in rats in vitro and in vivo. Animal experiment was a randomized, three-period crossover design. Significant changes in pharmacokinetic parameters of CZN such as Cmax, t1/2 and Vd were observed after treatment with baicalin in vivo (P<0.05). Cmax decreased by 25% and 33%, whereas t1/2 increased by 34% and 53%, Vd increased by 37% and 50% in 225 mg/kg and 450 mg/kg baicalin-treated rats, respectively. The AUC and CL of CZN were not affected (P>0.05). Correlation analysis showed that the changes in CZN concentrations and baicalin concentrations were in good correlation (r>0.99). In vitro experiments, baicalin decreased the formation of 6-OH-chlorzoxazone in a concentration-dependent manner and exhibited a competitive inhibition in rat liver microsomes, with a Ki value of 145.8 µM. The values of Cmax/Ki were 20 and 39 after treatment with baicalin (225 and 450 mg/kg), respectively. Protein binding experiments in vivo showed that the plasma free-fraction (fu) of CZN increased 2.6-fold immediately after baicalin treatment (450 mg/kg) and in vitro showed that baicalin (125–2500 mg/L) increased the unbound CZN from 1.63% to 3.58%. The results indicate that pharmacokinetic changes in CZN are induced by inhibitory effect of baicalin on the plasma protein binding of CZN and CYP2E1 activity. PMID:23301016

Assessment and characterisation of yeast-based products intended to mitigate ochratoxin exposure using in vitro and in vivo models.

PubMed

Pfohl-Leszkowicz, A; Hadjeba-Medjdoub, K; Ballet, N; Schrickx, J; Fink-Gremmels, J

2015-01-01

The aim of this paper was to evaluate the capacity of several yeast-based products, derived from baker's and brewer's yeasts, to sequester the mycotoxin ochratoxin A (OTA) and to decrease its rate of absorption and DNA adduct formation in vivo. The experimental protocol included in vitro binding studies using isotherm models, in vivo chicken experiments, in which the serum and tissue concentrations of OTA were analysed in the absence and presence of the test compounds, and the profile of OTA-derived metabolites and their associated DNA adducts were determined. Additionally in vitro cell culture studies (HK2 cells) were applied to assess further the effects for yeast cell product enriched with glutathione (GSH) or selenium. Results of the in vitro binding assay in a buffer system indicated the ability of the yeast-based products, as sequester of OTA, albeit at a different level. In the in vitro experiments in chickens, decreased serum and tissue concentrations of treated animals confirmed that yeast-based products are able to prevent the absorption of OTA. A comparison of the binding affinity in a standard in vitro binding assay with the results obtained in an in vivo chicken experiment, however, showed a poor correlation and resulted in a different ranking of the products. More importantly, we could show that yeast-based products actively modulate the biotransformation of OTA in vivo as well as in vitro in a cell culture model. This effect seems to be attributable to residual enzymatic activities in the yeast-based products. An enrichment of yeast cell wall products with GSH or selenium further modulated the profile of the generated OTA metabolites and the associated pattern of OTA-induced DNA adducts by increasing the conversion of OTA into less toxic metabolites such as OTA, OTB and 4-OH-OTA. A reduced absorption and DNA adduct formation was particularly observed with GSH-enriched yeast, whereas selenium-enriched yeasts could counteract the OTA-induced decrease in cell viability, but at the same time increased the OTA-DNA adducts formation. These findings indicate the need for an in-depth characterisation of yeast-based products used as mycotoxin-mitigating feed additives, in in vivo models with target animal species taking into account not only their ability to sequester toxins in the gastrointestinal tract but also their potential effects on the biotransformation of mycotoxins.

Silibinin and indocyanine green-loaded nanoparticles inhibit the growth and metastasis of mammalian breast cancer cells in vitro.

PubMed

Sun, Hui-Ping; Su, Jing-Han; Meng, Qing-Shuo; Yin, Qi; Zhang, Zhi-Wen; Yu, Hai-Jun; Zhang, Peng-Cheng; Wang, Si-Ling; Li, Ya-Ping

2016-07-01

To improve the therapeutic efficacy of cancer treatments, combinational therapies based on nanosized drug delivery system (NDDS) has been developed recently. In this study we designed a new NDDS loaded with an anti-metastatic drug silibinin and a photothermal agent indocyanine green (ICG), and investigated its effects on the growth and metastasis of breast cancer cells in vitro. Silibinin and ICG were self-assembled into PCL lipid nanoparticles (SIPNs). Their physical characteristics including the particle size, zeta potential, morphology and in vitro drug release were examined. 4T1 mammalian breast cancer cells were used to evaluate their cellular internalization, cytotoxicity, and their influences on wound healing, in vitro cell migration and invasion. SIPNs showed a well-defined spherical shape with averaged size of 126.3±0.4 nm and zeta potential of -10.3±0.2 mV. NIR laser irradiation substantially increased the in vitro release of silibinin from the SIPNs (58.3% at the first 8 h, and 97.8% for the total release). Furthermore, NIR laser irradiation markedly increased the uptake of SIPNs into 4T1 cells. Under the NIR laser irradiation, both SIPNs and IPNs (PCL lipid nanoparticles loaded with ICG alone) caused dose-dependent ablation of 4T1 cells. The wound healing, migration and invasion experiments showed that SIPNs exposed to NIR laser irradiation exhibited dramatic in vitro anti-metastasis effects. SIPNs show temperature-sensitive drug release following NIR laser irradiation, which can inhibit the growth and metastasis of breast cancer cells in vitro.

Active locomotion of a paddling-based capsule endoscope in an in vitro and in vivo experiment (with videos).

PubMed

Kim, Hee Man; Yang, Sungwook; Kim, Jinseok; Park, Semi; Cho, Jae Hee; Park, Jeong Youp; Kim, Tae Song; Yoon, Eui-Sung; Song, Si Young; Bang, Seungmin

2010-08-01

Capsule endoscopy that could actively move and approach a specific site might be more valuable for the diagnosis or treatment of GI diseases. We tested the performance of active locomotion of a novel wired capsule endoscope with a paddling-based locomotion mechanism, using 3 models: a silicone tube, an extracted porcine colon, and a living pig. In vitro, ex vivo, and in vivo experiments in a pig model. Study in an animal laboratory. For the in vitro test, the locomotive capsule was controlled to actively move from one side of a silicone tube to the other by a controller-operated automatic traveling program. The velocity was calculated by following a video recording. We performed ex vivo tests by using an extracted porcine colon in the same manner we performed the in vitro test. In in vivo experiments, the capsule was inserted into the rectum of a living pig under anesthesia, and was controlled to move automatically forward. After 8 consecutive trials, the velocity was calculated. Elapsed time, velocity, and mucosal damage. The locomotive capsule showed stable and active movement inside the lumen both in vitro and ex vivo. The velocity was 60 cm/min in the silicone tube, and 36.8 and 37.5 cm/min in the extracted porcine colon. In the in vivo experiments, the capsule stably moved forward inside the colon of a living pig without any serious complications. The mean velocity was 17 cm/min over 40 cm length. We noted pinpoint erythematous mucosal injuries in the colon. Porcine model experiments, wired capsule endoscope. The novel paddling-based locomotive capsule endoscope performed fast and stable movement in a living pig colon with consistent velocity. Further investigation is necessary for practical use in humans. Copyright 2010 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.

Purification and identification of a polysaccharide from medicinal mushroom Amauroderma rude with immunomodulatory activity and inhibitory effect on tumor growth.

PubMed

Pan, Honghui; Han, Yuanyuan; Huang, Jiguo; Yu, Xiongtao; Jiao, Chunwei; Yang, Xiaobing; Dhaliwal, Preet; Xie, Yizhen; Yang, Burton B

2015-07-10

Medicinal mushrooms in recent years have been the subject of many experiments searching for anticancer properties. We previously screened thirteen mushrooms for their potential in inhibiting tumor growth, and found that the water extract of Amauroderma rude exerted the highest activity. Previous studies have shown that the polysaccharides contained in the water extract were responsible for the anticancer properties. This study was designed to explore the potential effects of the polysaccharides on immune regulation and tumor growth. Using the crude Amauroderma rude extract, in vitro experiments showed that the capacities of spleen lymphocytes, macrophages, and natural killer cells were all increased. In vivo experiments showed that the extract increased macrophage metabolism, lymphocyte proliferation, and antibody production. In addition, the partially purified product stimulated the secretion of cytokines in vitro, and in vivo. Overall, the extract decreased tumor growth rates. Lastly, the active compound was purified and identified as polysaccharide F212. Most importantly, the purified polysaccharide had the highest activity in increasing lymphocyte proliferation. In summary, this molecule may serve as a lead compound for drug development.

Baicalin, a metabolite of baicalein with antiviral activity against dengue virus

PubMed Central

Moghaddam, Ehsan; Teoh, Boon-Teong; Sam, Sing-Sin; Lani, Rafidah; Hassandarvish, Pouya; Chik, Zamri; Yueh, Andrew; Abubakar, Sazaly; Zandi, Keivan

2014-01-01

Baicalin, a flavonoid derived from Scutellaria baicalensis, is the main metabolite of baicalein released following administration in different animal models and human. We previously reported the antiviral activity of baicalein against dengue virus (DENV). Here, we examined the anti-DENV properties of baicalin in vitro, and described the inhibitory potentials of baicalin at different steps of DENV-2 (NGC strain) replication. Our in vitro antiviral experiments showed that baicalin inhibited virus replication at IC50 = 13.5 ± 0.08 μg/ml with SI = 21.5 following virus internalization by Vero cells. Baicalin exhibited virucidal activity against DENV-2 extracellular particles at IC50 = 8.74 ± 0.08 μg/ml and showed anti-adsorption effect with IC50 = 18.07 ± 0.2 μg/ml. Our findings showed that baicalin as the main metabolite of baicalein exerting in vitro anti-DENV activity. Further investigations on baicalein and baicalin to deduce its antiviral therapeutic effects are warranted. PMID:24965553

Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers.

PubMed

Siril, E A; Joseph, Nisha

2013-01-01

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 μM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 μM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.

In vitro fertilization experiments using sockeye salmon reveal that bigger eggs are more fertilizable under sperm limitation

PubMed Central

Macfarlane, Christopher P.; Hoysak, Drew J.; Liley, N. Robin; Gage, Matthew J.G.

2009-01-01

Although theory and widespread evidence show that the evolution of egg size is driven primarily by offspring and maternal fitness demands, an additional explanation invokes sperm limitation as a selective force that could also influence egg size optima. Levitan proposed that constraints from gamete encounter in external fertilization environments could select for enlargement of ova to increase the physical size of the fertilization target. We test this theory using in vitro fertilization experiments in an externally fertilizing fish. Sockeye salmon (Onchorhyncus nerka) females show considerable between-individual variation in ovum size, and we explored the consequences of this natural variation for the fertilization success of individual eggs under conditions of sperm limitation. By engineering consistent conditions where in vitro fertilization rate was always intermediate, we were able to compare the sizes of fertilized and unfertilized eggs across 20 fertilization replicates. After controlling for any changes in volume through incubation, results showed that successfully fertilized eggs were significantly larger than the eggs that failed to achieve fertilization. Under conditions without sperm limitation, fertility was unaffected by egg size. Our findings therefore support Levitan's theory, demonstrating empirically that some element of egg size variation could be selected by fertilization demands under sperm limitation. However, further research on sperm limitation in natural spawnings is required to assess the selective importance of these results. PMID:19364734

In vitro fertilization experiments using sockeye salmon reveal that bigger eggs are more fertilizable under sperm limitation.

PubMed

Macfarlane, Christopher P; Hoysak, Drew J; Liley, N Robin; Gage, Matthew J G

2009-07-07

Although theory and widespread evidence show that the evolution of egg size is driven primarily by offspring and maternal fitness demands, an additional explanation invokes sperm limitation as a selective force that could also influence egg size optima. Levitan proposed that constraints from gamete encounter in external fertilization environments could select for enlargement of ova to increase the physical size of the fertilization target. We test this theory using in vitro fertilization experiments in an externally fertilizing fish. Sockeye salmon (Onchorhyncus nerka) females show considerable between-individual variation in ovum size, and we explored the consequences of this natural variation for the fertilization success of individual eggs under conditions of sperm limitation. By engineering consistent conditions where in vitro fertilization rate was always intermediate, we were able to compare the sizes of fertilized and unfertilized eggs across 20 fertilization replicates. After controlling for any changes in volume through incubation, results showed that successfully fertilized eggs were significantly larger than the eggs that failed to achieve fertilization. Under conditions without sperm limitation, fertility was unaffected by egg size. Our findings therefore support Levitan's theory, demonstrating empirically that some element of egg size variation could be selected by fertilization demands under sperm limitation. However, further research on sperm limitation in natural spawnings is required to assess the selective importance of these results.

Polyketide synthases from poison hemlock (Conium maculatum L.).

PubMed

Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

2015-11-01

Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. © 2015 FEBS.

Rational Design of a New Class of Toll-Like Receptor 4 (TLR4) Tryptamine Related Agonists by Means of the Structure- and Ligand-Based Virtual Screening for Vaccine Adjuvant Discovery.

PubMed

Honegr, Jan; Dolezal, Rafael; Malinak, David; Benkova, Marketa; Soukup, Ondrej; Almeida, Joyce S F D de; Franca, Tanos C C; Kuca, Kamil; Prymula, Roman

2018-01-04

In order to identify novel lead structures for human toll-like receptor 4 ( h TLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the h TLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate h TLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6.

Animal experimental studies using small intestine endoscope

PubMed Central

Liu, Jin-Hua; Liu, Dan-Yang; Wang, Li; Han, Li-Ping; Qi, Zhe-Yu; Ren, Hai-Jun; Feng, Yan; Luan, Feng-Ming; Mi, Liang-Tian; Shan, Shu-Mei

2017-01-01

AIM To assess the feasibility and safety of a novel enteroscope, negative-pressure suction endoscope in examining the small intestine of a porcine model. METHODS In vitro experiments in small intestinal loops from 20 pigs and in vivo experiments in 20 living pigs were conducted. RESULTS In in vitro experiments, a negative pressure of > 0.06 MPa was necessary for optimal visualization of the intestine, and this pressure did not cause gross or histological damage to the mucosa. For satisfactory examination of the small intestine in vivo, higher negative pressure (> 1.00 MPa) was required. Despite this higher pressure, the small intestine did not show any gross or microscopic damage in the suctioned areas. The average time of examination in the living animals was 60 ± 7.67 min. The animals did not experience any apparent ill effects from the procedure. CONCLUSION Small intestine endoscope was safely performed within a reasonable time period and enabled complete visualization of the intestine in most cases. PMID:28611521

Cross-study projections of genomic biomarkers: an evaluation in cancer genomics.

PubMed

Lucas, Joseph E; Carvalho, Carlos M; Chen, Julia Ling-Yu; Chi, Jen-Tsan; West, Mike

2009-01-01

Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a "common currency" that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies--in cancer and other diseases--have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes.

In vitro and in vivo Efficacy of New Blue Light Emitting Diode Phototherapy Compared to Conventional Halogen Quartz Phototherapy for Neonatal Jaundice

PubMed Central

Chang, Yun Sil; Hwang, Jong Hee; Kwon, Hyuk Nam; Choi, Chang Won; Ko, Sun Young; Park, Won Soon; Shin, Son Moon

2005-01-01

High intensity light emitting diodes (LEDs) are being studied as possible light sources for the phototherapy of neonatal jaundice, as they can emit high intensity light of narrow wavelength band in the blue region of the visible light spectrum corresponding to the spectrum of maximal bilirubin absorption. We developed a prototype blue gallium nitride LED phototherapy unit with high intensity, and compared its efficacy to commercially used halogen quartz phototherapy device by measuring both in vitro and in vivo bilirubin photodegradation. The prototype device with two focused arrays, each with 500 blue LEDs, generated greater irradiance than the conventional device tested. The LED device showed a significantly higher efficacy of bilirubin photodegradation than the conventional phototherapy in both in vitro experiment using microhematocrit tubes (44±7% vs. 35±2%) and in vivo experiment using Gunn rats (30±9% vs. 16±8%). We conclude that high intensity blue LED device was much more effective than conventional phototherapy of both in vitro and in vivo bilirubin photodegradation. Further studies will be necessary to prove its clinical efficacy. PMID:15716604

Preparation and in vitro/in vivo evaluation of gestodene (GEST) intravaginal ring.

PubMed

Li, Chunxiao; Ning, Meiying; Yao, Xiaodong; Wang, Yankun; Liu, Ying

2016-09-01

Preparation and in vitro/in vivo evaluation of gestodene (GEST) intravaginal ring (IVR) formulations which can release a constant dose of GEST during 3 weeks were investigated. In present study a reservoir gestodene intravaginal ring, including a gestodene silicone elastomer core and the non-active silicone layer, was reported, which was manufactured by reaction injection moulding at 80°C for 20 min. The raw materials compatibility experiments showed that the silicone elastomer core carrier wouldn't interact with drugs. In vitro release samples were determined by HPLC and the experiment was performed under sink conditions. The equation of cumulative release verse time was Y=64.76χ+5.44 (r=0.9998), performing zero-order release at about the target dose of 60 µg/day over 21 days. Drug release increased with temperature elevating from 45 to 55°C, which could be attributed to optimizing the prescription. In addition, the pharmacokinetic and safety studies of gestodene intravaginal ring were evaluated in female New Zealand White rabbits. The GEST in plasma was analyzed by LC-MS/MS and the results proved that the correlation between in vitro and in vivo was relatively well.

Radiolabeled cholesteryl ethers: A need to analyze for biological stability before use.

PubMed

Manual Kollareth, Denny Joseph; Chang, Chuchun L; Hansen, Inge H; Deckelbaum, Richard J

2018-03-01

Radiolabeled cholesteryl ethers are widely used as non-metabolizable tracers for lipoproteins and lipid emulsions in a variety of in vitro and in vivo experiments. Since cholesteryl ethers do not leave cells after uptake and are not hydrolyzed by mammalian cellular enzymes, these compounds can act as markers for cumulative cell uptakes of labeled particles. We have employed [ 3 H]cholesteryl oleoyl ether to study the uptake and distribution of triglyceride-rich emulsion particles on animal models. However, questionable unexpected results compelled us to analyze the stability of these ethers. We tested the stability of two commercially available radiolabeled cholesteryl ethers - [ 3 H]cholesteryl oleoyl ether and [ 3 H]cholesteryl hexadecyl ether from different suppliers, employing in vitro , in vivo and chemical model systems. Our results show that, among the two cholesteryl ethers tested, one ether was hydrolyzed to free cholesterol in vitro , in vivo and chemically under alkaline hydrolyzing agent. Free cholesterol, unlike cholesteryl ether, can then re-enter the circulation leading to confounding results. The other ether was not hydrolyzed to free cholesterol and remained as a stable ether. Hence, radiolabeled cholesteryl ethers should be analyzed for biological stability before utilizing them for in vitro or in vivo experiments.

Surface modification of nano-silica on the ligament advanced reinforcement system for accelerated bone formation: primary human osteoblasts testing in vitro and animal testing in vivo

NASA Astrophysics Data System (ADS)

Li, Mengmeng; Wang, Shiwen; Jiang, Jia; Sun, Jiashu; Li, Yuzhuo; Huang, Deyong; Long, Yun-Ze; Zheng, Wenfu; Chen, Shiyi; Jiang, Xingyu

2015-04-01

The Ligament Advanced Reinforcement System (LARS) has been considered as a promising graft for ligament reconstruction. To improve its biocompatibility and effectiveness on new bone formation, we modified the surface of a polyethylene terephthalate (PET) ligament with nanoscale silica using atom transfer radical polymerization (ATRP) and silica polymerization. The modified ligament is tested by both in vitro and in vivo experiments. Human osteoblast testing in vitro exhibits an ~21% higher value in cell viability for silica-modified grafts compared with original grafts. Animal testing in vivo shows that there is new formed bone in the case of a nanoscale silica-coated ligament. These results demonstrate that our approach for nanoscale silica surface modification on LARS could be potentially applied for ligament reconstruction.The Ligament Advanced Reinforcement System (LARS) has been considered as a promising graft for ligament reconstruction. To improve its biocompatibility and effectiveness on new bone formation, we modified the surface of a polyethylene terephthalate (PET) ligament with nanoscale silica using atom transfer radical polymerization (ATRP) and silica polymerization. The modified ligament is tested by both in vitro and in vivo experiments. Human osteoblast testing in vitro exhibits an ~21% higher value in cell viability for silica-modified grafts compared with original grafts. Animal testing in vivo shows that there is new formed bone in the case of a nanoscale silica-coated ligament. These results demonstrate that our approach for nanoscale silica surface modification on LARS could be potentially applied for ligament reconstruction. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01439e

EGAR, A Food Protein-Derived Tetrapeptide, Reduces Seizure Activity in Pentylenetetrazole-Induced Epilepsy Models Through α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionate Receptors.

PubMed

Cai, Song; Ling, Chuwen; Lu, Jun; Duan, Songwei; Wang, Yingzhao; Zhu, Huining; Lin, Ruibang; Chen, Liang; Pan, Xingchang; Cai, Muyi; Gu, Huaiyu

2017-01-01

A primary pathogeny of epilepsy is excessive activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs). To find potential molecules to inhibit AMPARs, high-throughput screening was performed in a library of tetrapeptides in silico. Computational results suggest that some tetrapeptides bind stably to the AMPAR. We aligned these sequences of tetrapeptide candidates with those from in vitro digestion of the trout skin protein. Among salmon-derived products, Glu-Gly-Ala-Arg (EGAR) showed a high biological affinity toward AMPAR when tested in silico. Accordingly, natural EGAR was hypothesized to have anticonvulsant activity, and in vitro experiments showed that EGAR selectively inhibited AMPAR-mediated synaptic transmission without affecting the electrophysiological properties of hippocampal pyramidal neurons. In addition, EGAR reduced neuronal spiking in an in vitro seizure model. Moreover, the ability of EGAR to reduce seizures was evaluated in a rodent epilepsy model. Briefer and less severe seizures versus controls were shown after mice were treated with EGAR. In conclusion, the promising experimental results suggest that EGAR inhibitor against AMPARs may be a target for antiepilepsy pharmaceuticals. Epilepsy is a common brain disorder characterized by the occurrence of recurring, unprovoked seizures. Twenty to 30 % of persons with epilepsy do not achieve adequate seizure control with any drug. Here we provide a possibility in which a natural and edible tetrapeptide, EGAR, can act as an antiepileptic agent. We have combined computation with in vitro experiments to show how EGAR modulates epilepsy. We also used an animal model of epilepsy to prove that EGAR can inhibit seizures in vivo. This study suggests EGAR as a potential pharmaceutical for the treatment of epilepsy.

Cytotoxic activity of natural killer cells in vitro under microgravity

NASA Astrophysics Data System (ADS)

Grigorieva, O. V.; Buravkova, L. B.; Rykova, M. P.

2005-08-01

Changes in the immune response during space flight are close relation to functions of NK lymphocytes and their ability to interact with target cells. The aim of this research was to study NK cells cytotoxic activity and their ability to produce cytokines under microgravity in vitro. The modification of the method to study NK cells cytotoxic activity with the use of human peripheral blood mononuclear cells and myeloblasts K-562 (as target cells) proved highly effective (Buravkova et al., 2004). The flight experiment "Cell-to-cell interaction" with the use of the special device "Fibroblast-1" was carried out by Russian cosmonauts within the first two days after the docking when a new crew was taking over on International Space Station (ISS 8 - 10). The data collected on board ISS revealed that NK lymphocytes cytotoxic activity in vitro can increase under microgravity. The ground-based simulation experiments showed that long-term changes in gravity vector direction clinorotation resulted in a smaller increase of NK cells cytotoxic activity than it did in microgravity. As lymphocytes produce cytokines while interacting with target cells, the levels of TNF-α, IL-1α, IL- 2, IL-6 in cell-conditioned medium were assessed. The data showed that microgravity has varied effects on cytokines production level.

Acanthamoeba castellanii is not be an adequate model to study human adenovirus interactions with macrophagic cells

PubMed Central

Cateau, Estelle; Leveque, Nicolas; Kaaki, Sihem; Beby-Defaux, Agnès; Rodier, Marie-Hélène

2017-01-01

Free living amoebae (FLA) including Acanthamoeba castellanii, are protozoa that feed on different microorganisms including viruses. These microorganisms show remarkable similarities with macrophages in cellular structures, physiology or ability to phagocyte preys, and some authors have therefore wondered whether Acanthamoeba and macrophages are evolutionary related. It has been considered that this amoeba may be an in vitro model to investigate relationships between pathogens and macrophagic cells. So, we intended in this study to compare the interactions between a human adenovirus strain and A. castellanii or THP-1 macrophagic cells. The results of molecular and microscopy techniques following co-cultures experiments have shown that the presence of the adenovirus decreased the viability of macrophages, while it has no effect on amoebic viability. On another hand, the viral replication occurred only in macrophages. These results showed that this amoebal model is not relevant to explore the relationships between adenoviruses and macrophages in in vitro experiments. PMID:28591183

MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo.

PubMed

Torella, Daniele; Iaconetti, Claudio; Catalucci, Daniele; Ellison, Georgina M; Leone, Angelo; Waring, Cheryl D; Bochicchio, Angela; Vicinanza, Carla; Aquila, Iolanda; Curcio, Antonio; Condorelli, Gianluigi; Indolfi, Ciro

2011-09-30

MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal-regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Activity of antibacterial, antiviral, anti-inflammatory in compounds andrographolide salt.

PubMed

Wen, Li; Xia, Nan; Chen, Xianghong; Li, Yingxiu; Hong, Yi; Liu, YaJie; Wang, ZiZhen; Liu, YaJie

2014-10-05

Andrographolide sulfonic acid sodium salt (ASS) was synthesized to increase the the solubility of Andrographolide in aqueous solution. We have studied its pharmacological effect of antibiosis, anti-inflammatory and immunoregulation. Cylinder-plate method was used to study ASS׳s in vitro antibacterial activity, and its protection for mice infected by Staphylococcus aureus and Shigella dysenteriae. Various inflammation models, including the auricular edema induced by xylene in mice, CMC-Na induced air pounch model and the paw edema induced by albumen in rats were used to explore the characteristic of ASS׳s anti-inflammation effect. We built up the immune model by injecting chicken red cells in enter celiac of mice and study the effect of ASS on immunoregulation, taking andrographolide as the positive control. bacteriostasis in vivo and in vitro experiments show that ASS has a weak antibacterial effect and no bactericidal effect, but can reduce the mice mortality of Staphylococcus aureus infected. Anti-inflammatory experiments show that ASS can reduce the mouse ear swelling induced by xylene and rat paw swelling induced by egg albumin, and lessen leukocytes in air bag caused by CMCNa, and lower IL1 not ably in rat serum. Immune tests indicate that ASS can get spleen and thymus gain weight and increase rate of abdominal macrophage phagocytosis of mice. The result of bacteriostasis shows that ASS has weak in vitro antibacterial effect. ASS shows significant effects of anti-inflammation and improving immunity, thus enables the mice against bacteria better. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterizing parameters of Jatropha curcas cell cultures for microgravity studies

NASA Astrophysics Data System (ADS)

Vendrame, Wagner A.; Pinares, Ania

2013-06-01

Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters evaluated in this study provide the basic ground work and pre-flight assessment needed to justify a model for microgravity studies with jatropha in vitro cell cultures. Future studies should focus on results of experiments performed with jatropha in vitro cultures in microgravity.

Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics.

PubMed

Rawat, Preeti; Singh, Amarjeet Kumar; Ray, Krishna; Chaudhary, Bhupendra; Kumar, Sanjeev; Gautam, Taru; Kanoria, Shaveta; Kaur, Gurpreet; Kumar, Paritosh; Pental, Deepak; Burma, Pradeep Kumar

2011-06-01

High levels of expression of the cry1Ac gene from Bacillus thuringiensis cannot be routinely achieved in transgenic plants despite modifications made in the gene to improve its expression. This has been attributed to the instability of the transcript in a few reports. In the present study, based on the genetic transformation of cotton and tobacco, we show that the expression of the Cry1Ac endotoxin has detrimental effects on both the in vitro and in vivo growth and development of transgenic plants. A number of experiments on developing transgenics in cotton with different versions of cry1Ac gene showed that the majority of the plants did not express any Cry1Ac protein. Based on Southern blot analysis, it was also observed that a substantial number of lines did not contain the cry1Ac gene cassette although they contained the marker gene nptII. More significantly, all the lines that showed appreciable levels of expression were found to be phenotypically abnormal. Experiments on transformation of tobacco with different constructs expressing the cry1Ac gene showed that in vitro regeneration was inhibited by the encoded protein. Further, out of a total of 145 independent events generated with the different cry1Ac gene constructs in tobacco, only 21 showed expression of the Cry1Ac protein, confirming observations made in cotton that regenerants that express high levels of the Cry1Ac protein are selected against during regeneration of transformed events. This problem was circumvented by targeting the Cry1Ac protein to the chloroplast, which also significantly improved the expression of the protein.

In vitro molecular machine learning algorithm via symmetric internal loops of DNA.

PubMed

Lee, Ji-Hoon; Lee, Seung Hwan; Baek, Christina; Chun, Hyosun; Ryu, Je-Hwan; Kim, Jin-Woo; Deaton, Russell; Zhang, Byoung-Tak

2017-08-01

Programmable biomolecules, such as DNA strands, deoxyribozymes, and restriction enzymes, have been used to solve computational problems, construct large-scale logic circuits, and program simple molecular games. Although studies have shown the potential of molecular computing, the capability of computational learning with DNA molecules, i.e., molecular machine learning, has yet to be experimentally verified. Here, we present a novel molecular learning in vitro model in which symmetric internal loops of double-stranded DNA are exploited to measure the differences between training instances, thus enabling the molecules to learn from small errors. The model was evaluated on a data set of twenty dialogue sentences obtained from the television shows Friends and Prison Break. The wet DNA-computing experiments confirmed that the molecular learning machine was able to generalize the dialogue patterns of each show and successfully identify the show from which the sentences originated. The molecular machine learning model described here opens the way for solving machine learning problems in computer science and biology using in vitro molecular computing with the data encoded in DNA molecules. Copyright © 2017. Published by Elsevier B.V.

[Application research of DEI technique based on synchrotron X-ray source in imaging rabbit eyeball in vitro].

PubMed

Yin, Hong-xia; Huang, Zhi-feng; Wang, Zhen-chang; Liu, Zhao-hui; Li, Yong; Zhu, Pei-ping

2010-03-23

To study the application of DEI technique in imaging the small structures of rabbit eyeball. DEI technique was used to image the eyeball of New Zealand white rabbit in vitro. The experiments were performed using beamline 4W1A at the topography station of Beijing Synchrotron Radiation Facility (BSRF). DEI image showed clearly the fine structures of the rabbit eyeball, such as the transparent cornea, the sclera, the ciliaris, and the ciliary body. DEI is a new X-ray imaging modality which achieves high contrast and spatial resolution. It also showed obvious effect of edge enhancement. DEI has good potential in observing the micro-structures of eyeballs and other small organs.

Antagonistic activity of Bacillus subtilis SB1 and its biocontrol effect on tomato bacterial wilt

USDA-ARS?s Scientific Manuscript database

A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, isolated from tomato roots, showed a broad-spectrum of antimicrobial activity in in vitro experiments. It inhibited the growth of many plant pathogens, including Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, Fusarium ox...

Argentinean cultivars of Vitis vinifera grow better than European ones when cultured in vitro under salinity.

PubMed

Cavagnaro, Juan B; Ponce, María T; Guzmán, Javier; Cirrincione, Miguel A

2006-04-01

Argentinean Vitis vinifera cultivars although originated from Europe, have clear ampelographic and genotypic differences as compared with the European cultivars currently used in wine making. In vitro evaluation of salt tolerance has been used in many species. Our hypothesis was that Argentinean cultivars are more tolerant to salinity than European ones. Three European cultivars, Malbec, Cabernet Sauvignon and Chardonnay and four Argentincan cultivars, Cereza, Criolla Chica, Pedro Gimcnez and Torrontes Riojano were tested by in vitro culture. Treatments included: 1) Control, 2) 60 mEq/L of a mixture of three parts of NaCl and one part of CaCl2 and 3) 90 mEq/L of the salt mixture. Results from two experiments (I and II) are reported. No differences were found in plant survival, expressed as % of the respective control, among cultivars. Leaf area, leaf, stem and total dry matter (DM) in Experiment I and leaf area, leaf number and leaf, stem, root and total DM in Experiment II, were higher in Argentinean cultivars than in European ones. We conclude that Argentinean cultivars show better performance in growing under salinity, especially in the highest salt concentration. Differences among cultivars, inside each group, were found for most of the measured variables.

Loss of Optineurin In Vivo Results in Elevated Cell Death and Alters Axonal Trafficking Dynamics

PubMed Central

Paulus, Jeremiah D.; Link, Brian A.

2014-01-01

Mutations in Optineurin have been associated with ALS, glaucoma, and Paget’s disease of bone in humans, but little is known about how these mutations contribute to disease. Most of the cellular consequences of Optineurin loss have come from in vitro studies, and it remains unclear whether these same defects would be seen in vivo. To answer this question, we assessed the cellular consequences of Optineurin loss in zebrafish embryos to determine if they showed the same defects as have been described in the in vitro studies. We found that loss of Optineurin resulted in increased cell death, as well as subtle cell morphology, cell migration and vesicle trafficking defects. However, unlike experiments on cells in culture, we found no indication that the Golgi apparatus was disrupted or that NF-κB target genes were upregulated. Therefore, we conclude that in vivo loss of Optineurin shows some, but not all, of the defects seen in in vitro work. PMID:25329564

Synthesis, crystal structures, molecular docking, and in vitro biological activities evaluation of transition metal complexes with 4-(3,4-dichlorophenyl) piperazine-1-carboxylic acid

NASA Astrophysics Data System (ADS)

Chen, Zhi-Jian; Chen, Ya-Na; Xu, Chun-Na; Zhao, Shan-Shan; Cao, Qi-Yue; Qian, Shao-Song; Qin, Jie; Zhu, Hai-Liang

2016-08-01

Three novel mononuclear complexes, [MⅡ(L)2·2H2O], (M = Cu, Ni or Cd; HL = 4-(3,4-dichlorophenyl)piperazine-1-carboxylic acid)were synthesized and structurally determined by single-crystal X-ray diffraction. Molecular docking study preliminarily revealed that complex 1 had potential urease inhibitory activity. In accordance with the result of calculation, in vitro tests of the inhibitory activities of complexes 1-3 against jack bean urease showed complex 1 (IC50 = 8.17 ± 0.91 μM) had better inhibitory activities than the positive reference acetohydroxamic acid (AHA) (IC50 = 26.99 ± 1.43 μM), while complexes 2 and 3 showed no inhibitory activities., kinetics study was carried out to explore the mechanism of the inhibiting of the enzyme, and the result indicated that complex 1 was a competitive inhibitor of urease. Albumin binding experiment and in vitro toxicity evaluation of complex 1 were implemented to explore its Pharmacological properties.

Tolerogenic Dendritic Cells Generated by In Vitro Treatment With SAHA Are Not Stable In Vivo.

PubMed

Thewissen, Kristof; Broux, Bieke; Hendriks, Jerome J A; Vanhees, Mandy; Stinissen, Piet; Slaets, Helena; Hellings, Niels

2016-01-01

The aim of this study is to examine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can generate dendritic cells (DCs) with a stable tolerogenic phenotype to counteract autoimmune responses in an animal model of multiple sclerosis. We investigated if the tolerogenic potency of DCs could be increased by continuous treatment during in vitro differentiation toward DCs compared to standard 24-h in vitro treatment of already terminally differentiated DCs. We show that in vitro treatment with SAHA reduces the generation of new CD11c(+) DCs out of mouse bone marrow. SAHA-generated DCs show reduced antigen-presenting function as evidenced by a reduction in myelin endocytosis, a decreased MHC II expression, and a failure to upregulate costimulatory molecules upon LPS challenge. In addition, SAHA-generated DCs display a reduction in proinflammatory cytokines and molecules involved in apoptosis induction, inflammatory migration, and TLR signaling, and they are less immunostimulatory compared to untreated DCs. We demonstrated that the underlying mechanism involves a diminished STAT1 phosphorylation and was independent of STAT6 activation. Although in vitro results were promising, SAHA-generated DCs were not able to alleviate the development of experimental autoimmune encephalomyelitis in mice. In vitro washout experiments demonstrated that the tolerogenic phenotype of SAHA-treated DCs is reversible. Taken together, while SAHA potently boosts tolerogenic properties in DCs during the differentiation process in vitro, SAHA-generated DCs were unable to reduce autoimmunity in vivo. Our results imply that caution needs to be taken when developing DC-based therapies to induce tolerance in the context of autoimmune disease.

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

NASA Technical Reports Server (NTRS)

Tsang, Joyce; Joyce, Gerald F.

1996-01-01

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

Potential uses of terahertz pulse imaging in dentistry: caries and erosion detection

NASA Astrophysics Data System (ADS)

Longbottom, Christopher; Crawley, David A.; Cole, Bryan E.; Arnone, Donald D.; Wallace, Vincent P.; Pepper, Michael

2002-06-01

TeraHertz Pulse Imaging (TPI) is a relatively new imaging modality for medical and dental imaging. The aim of the present study was to make a preliminary assessment of the potential uses of TPI in clinical dentistry, particularly in relation to caries detection and the detection and monitoring of erosion. Images were obtained in vitro using a new TPI system developed by TeraView Ltd. We present data showing that TPI in vitro images of approximal surfaces of whole teeth demonstrate a distinctive shadowing in the presence of natural carious lesions in enamel. The thickness of this enamel shadowing appears to be related to lesion depth. The use of non-ionizing radiation to image such lesions non-destructively in vitro represents a significant step towards such measurements in vivo. In addition, data is presented which indicates that TPI may have a potential role in the detection and monitoring of enamel erosion. In vitro experiments on whole incisor teeth show that TPI is capable of detecting relatively small artificially induced changes in the buccal or palatal surface of the enamel of these teeth. Imaging of enamel thickness at such a resolution without ionizing radiation would represent a significant breakthrough if applicable in vivo.

Genotoxicity of tungsten carbide-cobalt (WC-Co) nanoparticles in vitro: mechanisms-of-action studies.

PubMed

Moche, Hélène; Chevalier, Dany; Vezin, Hervé; Claude, Nancy; Lorge, Elisabeth; Nesslany, Fabrice

2015-02-01

We showed previously that tungsten carbide-cobalt (WC-Co) nanoparticles (NP) can be used as a nanoparticulate positive control in some in vitro mammalian genotoxicity assays. Here, we investigate the mechanisms of action involved in WC-Co NP genotoxicity in L5178Y mouse lymphoma cells and primary human lymphocytes, in vitro. Data from the micronucleus assay coupled with centromere staining and from the chromosome-aberration assay show the involvement of both clastogenic and aneugenic events. Experiments with the formamidopyrimidine DNA glycosylase (FPG)-modified comet assay showed a slight (non-significant) increase in FPG-sensitive sites in the L5178Y mouse lymphoma cells but not in the human lymphocytes. Electron paramagnetic resonance spin-trapping results showed the presence of hydroxyl radicals (•OH) in WC-Co NP suspensions, with or without cells, but with time-dependent production in the presence of cells. However, a significant difference in •OH production was observed between human lymphocytes from two different donors. Using H2O2, we showed that WC-Co NP can participate in Fenton-like reactions. Thus, •OH might be produced either via intrinsic generation by WC-Co NP or through a Fenton-like reaction in the presence of cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhancing osseointegration of orthopedic implants with titania nanotube surfaces

NASA Astrophysics Data System (ADS)

Baker, Erin A.

Introduction: As joint arthroplasty surgical procedures increase annually, the development of new strategies, including novel materials and surface modifications, to attain solid bone-implant fixation are needed to increase implant terms of service. In this study, we evaluate two morphologies of titania nanotubes in both in vitro and in vivo experiments to quantify osseointegrative potential and material-level biocompatibility. Materials and Methods: Samples were prepared via an electrochemical etching process. Two different titania nanotube (TiNT) morphologies were produced, Aligned and Trabecular. For the in vitro experiment, Sprague Dawley (SD) rat marrow-derived bone marrow cells (BMC) were seeded on samples. Alkaline phosphatase (ALP) activity, osteocalcin (OC) expression, expression of relevant genes as well as cell attachment and morphology were assessed. In the first in vivo experiment, Kirschner wires were implanted unilaterally into SD rat femora with a TiNT-etched or unmodified (Control) implant. General health assessments and weekly body weights were recorded. At a 12-week endpoint, hematologic, systemic metal ion, and histologic analyses were performed. For the second in vivo experiment, Kirschner wires were implanted bilaterally into SD rat femora, with a TiNT-etched implant in one femora and unmodified (Control) implant as an internal control. At 4- and 12-week endpoints, femora were assessed via biomechanics, undecalcified histology, micro-computed tomography (muCT), and backscattered electron imaging (BEI) to characterize de novo bone formation. Results: In vitro experiments demonstrated BMC attachment and differentiation into osteoblasts as well as greater ALP activity, OC expression, total cell counts, and gene expression (of Col1a1, IGF-1, and osteonectin) on TiNT surfaces versus Controls. Cells on TiNT-etched substrates were smaller in diameter and more eccentric than Controls. In the first in vivo experiment, there were significant differences in body weight between groups at Weeks 9 and 11. There were no significant differences in red or white blood cell function between TiNT groups and Control. Aluminum levels in the lungs were significantly greater in the Trabecular TiNT group compared to Control. Histologic analysis showed significantly fewer granulocytes and neutrophils in the distal region of Trabecular TiNT-implanted femora as well as significantly fewer foreign body giant/multinucleated cells and neutrophils in the midshaft region of Aligned TiNT-implanted femora versus Controls. In the second in vivo experiment, at 12 weeks, microCT analysis showed TiNT implants generated greater bone formation than Controls. Histologic analysis demonstrated 1.5 times greater bone-implant contact in TiNT groups than Controls at 12 weeks. TiNT groups exhibited 1.3 to 3.7 times greater strength of fixation than Controls during pull-out testing. Discussion and Conclusions: In vitro data confirmed BMC attachment and differentiation into osteoblasts as well as osteoblastic phenotypic behavior. A clinically-relevant in vivo model of femoral intramedullary fixation, showed increased bone formation and quality in femora implanted with TiNT-etched implants versus Controls. A second in vivo study showed that TiNT surfaces do not generate systemic effects and may beneficially modulate the periprosthetic inflammatory environment.

Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Gardel, Margaret

Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field.

PubMed

Ding, Jing; Fu, Liang; Ding, Zhao-Wei; Lu, Yong-Ze; Cheng, Shuk H; Zeng, Raymond J

2016-01-01

The nitrate-dependent denitrifying anaerobic methane oxidation (DAMO) process, which is metabolized together by anaerobic methanotrophic archaea and NC10 phylum bacteria, is expected to be important for the global carbon and nitrogen cycles. However, there are little studies about the existence of this process and the functional microbes in environments. Therefore, the coexistence of DAMO archaea and bacteria in a paddy field was evaluated in this study. Next-generation sequencing showed that the two orders, Methanosarcinales and Nitrospirales, to which DAMO archaea and DAMO bacteria belong, were detected in the four soil samples. Then the in vitro experiments demonstrated both of nitrite- and nitrate-dependent DAMO activities, which confirmed the coexistence of DAMO archaea and DAMO bacteria. It was the first report about the coexistence of DAMO archaea and bacteria in a paddy field. Furthermore, anammox bacteria were detected in two of the four samples. The in vitro experiments did not show anammox activity in the initial period but showed low anammox activity after 20 days' enrichment. These results implicated that anammox bacteria may coexist with DAMO microorganisms in this field, but at a very low percentage.

Overexpression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo.

PubMed

Mahmoud, Salma; Ibrahim, Mohammed; Hago, Ahmed; Huang, Yuhong; Wei, Yuanyi; Zhang, Jun; Zhang, Qingqing; Xiao, Yu; Wang, Jingwen; Adam, Munkaila; Guo, Yu; Wang, Li; Zhou, Shuting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

2016-11-15

Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that up regulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 down regulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its over expression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy.

Overexpression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo

PubMed Central

Mahmoud, Salma; Ibrahim, Mohammed; Hago, Ahmed; Huang, Yuhong; Wei, Yuanyi; Zhang, Jun; Zhang, Qingqing; Xiao, Yu; Wang, Jingwen; Adam, Munkaila; Guo, Yu; Wang, Li; Zhou, Shuting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

2016-01-01

Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that upregulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 downregulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its overexpression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy. PMID:27626699

[Orthogonal experiments for optimizing the formulation and preparation conditions of temozolomide solid lipid nanoparticles].

PubMed

Dou, Mingjin; Huang, Guihua; Xi, Yanwei; Zhang, Na

2008-10-01

TMZ-SLN were prepared by emulsification-low temperature solidification method with stearic acid. The formulation and the preparation conditions were optimized by orthogonal experiments using entrapment efficiency as the evaluation index. The morphology was detected by transmission electron microscope. The Zeta potentials and the particle size distribution were evaluated by Laser Doppler Anemometry. The entrapment efficiencies and the drug release characteristics in vitro were assessed. The result showed that TMZ-SLN were concinnous and spherical in shape. The mean diameter (d(av) ) was 65.0 +/- 6.2 nm and the Zeta potential was -37.2 mV. The average entrapment efficiency was 58.9% +/- 1.21 %. The drug release behavior in vitro conformed to Higuchi Equation. The formation of a new material phase was testified by analysis of differential scanning calorimetry.

Corneal protection with high-molecular-weight hyaluronan against in vitro and in vivo sodium lauryl sulfate-induced toxic effects.

PubMed

Pauloin, Thierry; Dutot, Mélody; Liang, Hong; Chavinier, Emilie; Warnet, Jean-Michel; Rat, Patrice

2009-10-01

The aim of this study was to investigate high-molecular-weight hyaluronan (HA-HMW) corneal protection against sodium lauryl sulfate (SLS)-induced toxic effects with in vitro and in vivo experimental approaches. In vitro experiments consisted of a human corneal epithelial cell line incubated with HA-HMW, rinsed, and incubated with SLS. Cell viability, oxidative stress, chromatin condensation, caspase-3, -8, -9, and P2X7 cell death receptor activation, interleukin-6, and interleukin-8 production were investigated. In vivo experiments consisted of 36 New Zealand white rabbits treated for 3 days, 3 times per day, with HA-HMW or phosphate-buffered salt solution. At day 4, eyes were treated with SLS. Clinical observation and in vivo confocal microscopy using the Rostock Cornea Module of the Heidelberg Retina Tomograph-II were performed to evaluate and to compare SLS-induced toxicity between eyes treated with HA-HMW and eyes treated with phosphate-buffered salt solution. In vitro data indicate that exposure of human corneal epithelial cells to HA-HMW significantly decreased SLS-induced oxidative stress, apoptosis, and inflammation cytokine production. In vivo data indicate that SLS cornea injuries, characterized by damaged corneal epithelium, damaged anterior stroma, and inflammatory infiltrations, were attenuated with HA-HMW treatment. A good correlation was seen between in vitro and in vivo findings showing that HA-HMW decreases SLS-induced toxic effects and protects cornea.

Investigations on the in vitro bioactivity of swift heavy oxygen ion irradiated hydroxyapatite.

PubMed

Suganthi, R V; Prakash Parthiban, S; Elayaraja, K; Girija, E K; Kulariya, P; Katharria, Y S; Singh, F; Asokan, K; Kanjilal, D; Narayana Kalkura, S

2009-12-01

The effect of swift heavy oxygen ion irradiation of hydroxyapatite on its in vitro bioactivity was studied. The irradiation experiment was performed using oxygen ions at energy of 100 MeV with 1 x 10(12) and 1 x 10(13) ions/cm2 fluence range. The irradiated samples were characterized by glancing angle X-ray diffraction (GXRD), photoluminescence spectroscopy (PL) and scanning electron microscopy (SEM). GXRD showed that irradiated samples exhibited better crystallinity. The irradiated samples revealed an increase in PL intensity. In addition, the irradiated hydroxyapatite was found to have enhanced bioactivity.

Tilmicosin reduces lipopolysaccharide-stimulated bovine alveolar macrophage prostaglandin E(2) production via a mechanism involving phospholipases.

PubMed

Lakritz, Jeffrey; Tyler, Jeff W; Marsh, Antoinette E; Romesburg-Cockrell, Mary; Smith, Kathy; Holle, Julie M

2002-01-01

Tilmicosin is a potent antimicrobial with broad-spectrum activity against the bacterial agents involved in the bovine respiratory disease complex. Recent studies indicate that in addition to being bactericidal, tilmicosin is capable of modulating inflammation in the lung. A series of experiments were designed to determine whether tilmicosin alters alveolar macrophage-prostaglandin E(2) (PGE(2)) production induced by Escherichia coli (O55:B5) lipopolysaccharide (LPS). Twenty-two healthy Holstein bull calves were used to study the effects of LPS-induced PGE(2) production of alveolar macrophages after in vivo or in vitro treatment with tilmicosin. In Experiment 1, tilmicosin was given by subcutaneous injection (15 mg/kg) twice, 48 hours apart, to four calves; four control calves received no treatment. Twenty-four hours after the second treatment, alveolar macrophages were stimulated with LPS in vitro. In Experiment 2, alveolar macrophages from five untreated calves were harvested and treated in vitro with tilmicosin, followed by LPS stimulation. In Experiment 3, the ability of in vitro tilmicosin treatment to alter the expression of LPS-induced cyclooxygenase-2 (COX-2) mRNA was evaluated. In Experiments 4 and 5, secretory phospholipase A(2) activity was examined in untreated calves. Treatment of calves with tilmicosin resulted in reduced LPS-induced alveolar macrophage PGE(2) production. Similar reductions in PGE(2) by LPS-stimulated alveolar macrophages after in vitro tilmicosin treatment were noted. This in vitro tilmicosin treatment was not associated with reduction of the expression of LPS-induced COX-2. Alveolar macrophage phospholipase A(2) activity induced by LPS was significantly reduced by prior tilmicosin treatment in vitro. Tilmicosin (in vivo and in vitro) appears to reduce the PGE(2) eicosanoid response of LPS-stimulated alveolar macrophages by reducing the in vitro substrate availability without altering in vitro COX-2 mRNA expression.

Cholesterol supplementation improves growth rates of Histomonas meleagridis in vitro.

PubMed

Gruber, Janine; Pletzer, Alena; Hess, Michael

2018-02-01

Research on the energy metabolism of various protozoan parasites showed the essentiality and benefits of cholesterol in the cultivation of these organisms. However, not much is known about the energy metabolism of Histomonas meleagridis, although such information is of high importance to improve cultivation of the parasite for advancements in diagnostics, research and vaccine development. By supplementing a serum enriched cultivation medium with cholesterol, numbers of parasites could be doubled in comparison to unsupplemented negative controls. This effect was demonstrated for two different strains of the parasite, at different levels of in vitro-passages and for histomonads under xenic or monoxenic settings. Supplementing medium free of serum with cholesterol, resulted in significant growth of the parasite over 72 h. However, there were differences in growth behaviour in serum free medium between the different histomonad cultures and continuous passaging of the cultures without serum was not possible. Monitoring the bacterial growth of two different co-cultivated E. coli strains in monoxenic histomonad cultures during these experiments showed that there was no significant impact of cholesterol on the bacteria. Therefore, a direct effect of cholesterol on the parasite itself could be demonstrated. The results of these experiments supply new insights into the metabolism of H. meleagridis and it can be concluded that cholesterol is an important component to enhance parasite growth in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of raceme-localized supplemental light on soybean reproductive abscission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myers, R.L.; Brun, W.A.; Brenner, M.L.

The percentage of soybean (Glycine max (L.) Merr.) reproductive structures that abscise is a potentially important yield factor. To better understand the involvement of light in the abscission of reproductive structures, a series of in vitro raceme-culture and growth-chamber experiments were conducted. In the in vitro raceme-culture experiments, racemes with four to six flowers at or past anthesis were excised from the soybean plant (genotype IX93-100), embedded in a complete nutrient, solid agar medium, and subjected to various light treatments. A series of three experiments indicated that the racemes contain a photoreceptor, possibly phytochrome, capable of regulating sucrose accumulation. Inmore » each of the growth chamber studies, supplemental light was supplied directly to individual soybean flowers via fiber optic light guides. The light source increased the photon flux to the flowers by 10-fold. The first growth chamber experiment showed that flowers receiving supplemental light were more intense sinks for /sup 14/C-sucrose than were controls (intensity value of 1.0 vs 0.4 x 10/sup -7/, intensity = (dps of flower/dps of raceme)/(kg dry wt of flower)). In a second study, 42% of flowers treated with supplemental light set pods, while only 26% of control flowers set pods. A third experiment showed that red supplemental light produced 55% fruit set, compared to 41% set for far-red light, and 35% for controls. These experiments indicate that both photoassimilate accumulation and abscission in young soybean reproductive structures may be regulated by light quality.« less

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus.

PubMed

Hong, S K; Matsumoto, A; Horinouchi, S; Beppu, T

1993-01-01

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

A Novel Photosensitizer 3¹,13¹-phenylhydrazine -Mppa (BPHM) and Its in Vitro Photodynamic Therapy against HeLa Cells.

PubMed

Li, Wenting; Tan, Guanghui; Cheng, Jianjun; Zhao, Lishuang; Wang, Zhiqiang; Jin, Yingxue

2016-04-29

Photodynamic therapy (PDT) has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa) has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-3¹,13¹ bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT) against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT) assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm²). Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role.

Repair of pig dura in vivo using temperature controlled CO(2) laser soldering.

PubMed

Forer, Boaz; Vasilyev, Tamar; Brosh, Tamar; Kariv, Noam; Gil, Ziv; Fliss, Dan M; Katzir, Abraham

2005-10-01

The purpose of this study was to demonstrate that laser soldering might be successfully used for closing holes or cuts in the dura layer, which encapsulates the brain. A temperature controlled fiberoptic CO(2) laser system and albumin solder were used for spot soldering of fascia patches to holes in the dura of farm pigs, in vitro and in vivo. The mean burst pressure of the soldered patches in the in vitro experiments was 190 +/- 88 mm Hg-significantly higher than typical maximum CSF pressure of 15 mm Hg. In the in vivo experiments the pigs showed no postoperative complications. Histopathological studies exhibited an accepted level of inflammatory reaction and showed no thermal damage to the underlying brain tissue. It has been clearly demonstrated that temperature controlled laser soldering is a very useful technique for the repair of the dura. It provides significant advantages over standard closure techniques: it is easy to apply, the bond is strong and watertight and the procedure is likely to be much faster than suturing. This research work will lead to clinical trials.

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

NASA Astrophysics Data System (ADS)

Primo, Fernando L.; Rodrigues, Marcilene M. A.; Simioni, Andreza R.; Bentley, Maria V. L. B.; Morais, Paulo C.; Tedesco, Antonio C.

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quantification of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+ dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell's Franz device with receptor medium container with a PBS/EtOH 20% solution (10 mM, pH 7.4) at 37 °C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall.

Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions.

PubMed

Palmgrén, Joni J; Mönkkönen, Jukka; Korjamo, Timo; Hassinen, Anssi; Auriola, Seppo

2006-11-01

Loss of drug content during cell culture transport experiment can lead to misinterpretations in permeability analysis. This study analyses drug adsorption to various plastic containers and drug retention in cultured cells under in vitro conditions. The loss of various drugs to polystyrene tubes and well plates was compared to polypropylene and glass tubes both in deionised water and buffer solution. In cellular uptake experiments, administered drugs were obtained from cultured cells by liquid extraction. Samples were collected at various time points and drug concentrations were measured by a new HPLC-MS/MS method. Acidic drugs (hydrochlorothiazide, naproxen, probenicid, and indomethacin) showed little if any sorption to all tested materials in either water or buffer. In the case of basic drugs, substantial loss to polystyrene tubes and well plates was observed. After 4.5 h, the relative amount remaining in aqueous test solution stored in polystyrene tubes was 64.7 +/- 6.8%, 38.4 +/- 9.1%, 31.9 +/- 6.7%, and 23.5 +/- 6.1% for metoprolol, medetomidine, propranolol, and midazolam, respectively. Interestingly, there was no significant loss of drugs dissolved in buffer to any of the tested materials indicating that buffer reduced surficial interaction. The effect of drug concentration to sorption was also tested. Results indicated that the higher the concentration in the test solution the lower the proportional drug loss, suggesting that the polystyrene contained a limited amount of binding sites. Cellular uptake studies showed considerable retention of drugs in cultured cells. The amounts of absorbed drugs in cellular structures were 0.45%, 4.88%, 13.15%, 43.80%, 23.57% and 11.22% for atenolol, metoprolol, medetomidine, propranolol, midazolam, and diazepam, respectively. Overall, these findings will benefit development and validation of further in vitro drug permeation experiments.

Irisin protects against endothelial injury and ameliorates atherosclerosis in apolipoprotein E-Null diabetic mice.

PubMed

Lu, Junyan; Xiang, Guangda; Liu, Min; Mei, Wen; Xiang, Lin; Dong, Jing

2015-12-01

The circulating irisin increases energy expenditure and improves insulin resistance in mice and humans. The improvement of insulin resistance ameliorates atherosclerosis. Therefore, we hypothesized that irisin alleviates atherosclerosis in diabetes. Endothelial function was measured by acetylcholine-induced endothelium-dependent vasodilation using aortic rings in apolipoprotein E-Null (apoE(-/-)) streptozotocin-induced diabetic mice. Atherosclerotic lesion was evaluated by plaque area and inflammatory response in aortas. In addition, the endothelium-protective effects of irisin were also further investigated in primary human umbilical vein endothelial cells (HUVECs) in vitro. The in vivo experiments showed that irisin treatment significantly improved endothelial dysfunction, decreased endothelial apoptosis, and predominantly decreased atherosclerotic plaque area of both en face and cross sections when compared with normal saline-treated diabetic mice. Moreover, the infiltrating macrophages and T lymphocytes within plaque and the mRNA expression levels of inflammatory cytokines in aortas were also significantly reduced by irisin treatment in mice. The in vitro experiments revealed that irisin inhibited high glucose-induced apoptosis, oxidative stress and increased antioxidant enzymes expression in HUVECs, and pretreatment with LY294002, l-NAME, AMPK-siRNA or eNOS-siRNA, attenuated the protection of irisin on HUVECs apoptosis induced by high glucose. In addition, the in vivo and in vitro experiments showed that irisin increased the phosphorylation of AMPK, Akt and eNOS in aortas and cultured HUVECs. The present study indicates that systemic administration of irisin may be protected against endothelial injury and ameliorated atherosclerosis in apoE(-/-) diabetic mice. The endothelium-protective action of irisin was through activation of AMPK-PI3K-Akt-eNOS signaling pathway. Irisin could be therapeutic for atherosclerotic vascular diseases in diabetes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In vivo and in vitro efficacy of sainfoin (Onobrychis viciifolia) against Eimeria spp in lambs.

PubMed

Saratsis, Anastasios; Regos, Ionela; Tzanidakis, Nikolaos; Voutzourakis, Nikolaos; Stefanakis, Alexandros; Treuter, Dieter; Joachim, Anja; Sotiraki, Smaragda

2012-08-13

The effect of sainfoin (Onobrychis viciifolia) against ovine coccidia was evaluated in vivo and in vitro. In 3 in vivo trials weaned lambs were allocated into two treatment groups receiving diets with either lucerne (Medicago sativa) or sainfoin. During the trials, which lasted for 7 (trial 1) or 8 weeks (trials 2 and 3), oocysts per gram of faeces (OPGs), faecal scores and weight gain were recorded. In two of the experiments (trials 1 and 3) a reduction in the mean oocyst excretion rates was observed, starting three to four weeks after sainfoin hay feeding. This reduction ranged between 21.3% (trial 1) and 61.7% (trial 3) compared to the control values. As a result, a decrease in the total number of oocysts excreted (expressed as the mean area under the curve of the OPG) was observed from week 4 to the end of the two trials, respectively (trial 1: 42.6% reduction, p=0.05; trial 3: 52.4% reduction, p=0.06). The results did not show any significant diet effect on lamb growth rates and faecal scores. In the in vitro experiments the effect of 39 sainfoin extracts were tested in an oocyst sporulation inhibition assay. The Eimeria oocysts sporulation inhibition throughout the experiments did not exceed 10.7%, showing that extracts of this forages do not have a significant inhibitory effect on Eimeria oocyst sporulation. This was an initial attempt to investigate a possible anticoccidial effect of sainfoin and further studies are needed in order to better understand its mode of action against Eimeria. Copyright © 2012 Elsevier B.V. All rights reserved.

In-Vitro Immunology - Skylab Student Experiment ED-31

NASA Technical Reports Server (NTRS)

1973-01-01

This chart describes the Skylab student experiment In-Vitro Immunology, proposed by Todd A. Meister of Jackson Heights, New York. He suggested an in-vitro observation of the effects of zero-gravity on a presipitin-type antigen-antibody reaction, as compared with the same reaction carried out in an Earth-based laboratory. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

In vitro and in situ degradation of alkali treated sorghum wet distillers grains alone or in combination with corn stalks to increase their nutritive value

USDA-ARS?s Scientific Manuscript database

This experiment was conducted to evaluate the effect of alkali treatment on in vitro and in situ digestibility of fiber sources. An in vitro and in situ experiment were conducted to determine the effects of treating sorghum WDG with solubles (SWDG) and corn stalks (CS) with calcium hydroxide on in ...

One bis-indole alkaloid-voacamine from Voacanga africana Stapf: biological activity evaluation of PTP1B in vitro utilizing enzymology method based on SPRi expriment.

PubMed

Wang, Yan-Qiu; Li, Hong-Xiang; Liu, Xiao-Chun; Zhao, Jin-Shuang; Liu, Rong-Qiang; Huai, Wen-Ying; Ding, Wei-Jun; Zhang, Tian-E; Deng, Yun

2018-05-31

One known bis-indole alkaloid-voacamine was isolated from Voacanga africana Stapf and Surface Plasmon Resonance imaging (SPRi) exprement showed that this alkaloid could be combine with Protein Tyrosine Phosphatase1B (PTP1B). Then the PTP1B activity inhibition experiment display that the compound showed an outstanding promoting activity to PTP1B.

Monte Carlo simulations guided by imaging to predict the in vitro ranking of radiosensitizing nanoparticles

PubMed Central

Retif, Paul; Reinhard, Aurélie; Paquot, Héna; Jouan-Hureaux, Valérie; Chateau, Alicia; Sancey, Lucie; Barberi-Heyob, Muriel; Pinel, Sophie; Bastogne, Thierry

2016-01-01

This article addresses the in silico–in vitro prediction issue of organometallic nanoparticles (NPs)-based radiosensitization enhancement. The goal was to carry out computational experiments to quickly identify efficient nanostructures and then to preferentially select the most promising ones for the subsequent in vivo studies. To this aim, this interdisciplinary article introduces a new theoretical Monte Carlo computational ranking method and tests it using 3 different organometallic NPs in terms of size and composition. While the ranking predicted in a classical theoretical scenario did not fit the reference results at all, in contrast, we showed for the first time how our accelerated in silico virtual screening method, based on basic in vitro experimental data (which takes into account the NPs cell biodistribution), was able to predict a relevant ranking in accordance with in vitro clonogenic efficiency. This corroborates the pertinence of such a prior ranking method that could speed up the preclinical development of NPs in radiation therapy. PMID:27920524

Monte Carlo simulations guided by imaging to predict the in vitro ranking of radiosensitizing nanoparticles.

PubMed

Retif, Paul; Reinhard, Aurélie; Paquot, Héna; Jouan-Hureaux, Valérie; Chateau, Alicia; Sancey, Lucie; Barberi-Heyob, Muriel; Pinel, Sophie; Bastogne, Thierry

This article addresses the in silico-in vitro prediction issue of organometallic nanoparticles (NPs)-based radiosensitization enhancement. The goal was to carry out computational experiments to quickly identify efficient nanostructures and then to preferentially select the most promising ones for the subsequent in vivo studies. To this aim, this interdisciplinary article introduces a new theoretical Monte Carlo computational ranking method and tests it using 3 different organometallic NPs in terms of size and composition. While the ranking predicted in a classical theoretical scenario did not fit the reference results at all, in contrast, we showed for the first time how our accelerated in silico virtual screening method, based on basic in vitro experimental data (which takes into account the NPs cell biodistribution), was able to predict a relevant ranking in accordance with in vitro clonogenic efficiency. This corroborates the pertinence of such a prior ranking method that could speed up the preclinical development of NPs in radiation therapy.

Pursuing conception: a physician's experience with in-vitro fertilization.

PubMed Central

McCall, M

1996-01-01

Infertility is a common problem. Approximately one in seven North American couples will experience it, either by being unable to conceive after a year of trying or by experiencing recurrent miscarriages. A family physician outlines her experiences when being treated for infertility by in-vitro fertilization and embryo transfer. PMID:8625028

In vitro development of Haemoproteus parasites: the efficiency of reproductive cells increase during simultaneous sexual process of different lineages.

PubMed

Valkiūnas, Gediminas; Palinauskas, Vaidas; Ilgūnas, Mikas; Bernotienė, Rasa; Iezhova, Tatjana A

2014-04-01

Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporida) between-lineage interactions, which prevent mixing of lineages during simultaneous sexual process. Numerous anomalous ookinetes have been observed; these are not involved in sporogony. Massive development of such ookinetes might influence parasite transmission but is insufficiently investigated. The simultaneous sexual process of several lineages is a common phenomenon in vectors due to high prevalence of haemosporidian co-infections in wildlife. It remains unclear if the number of anomalous ookinetes changes during dual-infection sporogony in comparison with the single-infection process. We calculated proportions of the anomalous and normal ookinetes, which developed during single-infection (control) and dual-infection experiments in vitro conditions. Three mitochondrial cytochrome b lineages belonging to three Haemoproteus spp. (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes of two different parasites; the following experiments were performed: (1) Haemoproteus tartakovskyi (lineage hSISKIN1) × Haemoproteus lanii (lineage hRBS4) and (2) Haemoproteus belopolskyi (hHIICT3) × H. lanii (hRBS4). Genetic difference between lineages was 5.0-5.9%. Normal and anomalous ookinetes developed in all control and dual-infection experiments. The number of anomalous ookinetes markedly decreased, and normal ookinetes increased in all dual-infection experiments in comparison with those in controls, except for H. belopolskyi, in which proportion of the anomalous and normal ookinetes did not change. This study shows that simultaneous sexual process of two genetically distant lineages of haemosporidian parasites might increase the efficiency of reproductive cells, resulting in the development of a greater number of normal ookinetes. The marked increase of the number of normal ookinetes, which is involved in sporogony, indicates the success of sporogony in dual infections. Some haemosporidian lineages might benefit from simultaneous sporogony. Widespread avian Haemoproteus spp. are convenient and laboratory-friendly organisms for in vitro experimental research addressing between-lineage interaction in parasites during the sexual process.

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

PubMed

Mugnier, Sylvie; Kervella, Morgane; Douet, Cécile; Canepa, Sylvie; Pascal, Géraldine; Deleuze, Stefan; Duchamp, Guy; Monget, Philippe; Goudet, Ghylène

2009-11-19

Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes. In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates. Our study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

PubMed Central

2009-01-01

Background Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Methods & results In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes. In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates. Conclusion Our study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization. PMID:19925651

Low-frequency sonophoresis enhances rivastigmine permeation in vitro and in vivo.

PubMed

Yu, Zhen-wei; Liang, Yi; Liang, Wen-quan

2015-06-01

We investigated the enhancement effect of low-frequency sonophoresis on transdermal permeation of rivastigmine in vitro and in vivo. The in vitro permeation study showed that sonophoresis increased steady-state transdermal flux 0.31 ± 0.03 μg x cm(-2) x h(-1) and the extent of rivastigmine permeation 6.00 ± 0.56 μg x cm(-2) though excised skin (both P < 0.01). In the in vivo experiment, the C(max) 0.83 ± 0.16 μg x mL(-1) and AUC(0 --> 24 h) 12.35 ± 1.99 μg x h x mL(-1) of the sonophoresis group was also significantly higher than that of the control group (both P < 0.01). These data suggest that low-frequency sonophoresis could be an effective method to enhance rivastigmine permeation.

Surface modification of nano-silica on the ligament advanced reinforcement system for accelerated bone formation: primary human osteoblasts testing in vitro and animal testing in vivo.

PubMed

Li, Mengmeng; Wang, Shiwen; Jiang, Jia; Sun, Jiashu; Li, Yuzhuo; Huang, Deyong; Long, Yun-Ze; Zheng, Wenfu; Chen, Shiyi; Jiang, Xingyu

2015-05-07

The Ligament Advanced Reinforcement System (LARS) has been considered as a promising graft for ligament reconstruction. To improve its biocompatibility and effectiveness on new bone formation, we modified the surface of a polyethylene terephthalate (PET) ligament with nanoscale silica using atom transfer radical polymerization (ATRP) and silica polymerization. The modified ligament is tested by both in vitro and in vivo experiments. Human osteoblast testing in vitro exhibits an ∼21% higher value in cell viability for silica-modified grafts compared with original grafts. Animal testing in vivo shows that there is new formed bone in the case of a nanoscale silica-coated ligament. These results demonstrate that our approach for nanoscale silica surface modification on LARS could be potentially applied for ligament reconstruction.

In vivo and in vitro study of the function of the left and right bovine ovaries.

PubMed

Karamishabankareh, Hamed; Hajarian, Hadi; Shahsavari, Mohammadhamed; Moradinejad, Ruhollah

2015-09-15

Inequality in function of the left and right bovine ovaries and uterine horns was evaluated in two separate experiments. In the first experiment (in vivo), the relationship between the left and right ovarian activities and reproductive indices was evaluated. Therefore, the total number of 1284 randomly chosen lactating dairy cows were examined from Day 50 to 60 postpartum, and according to the presence of an active CL on the ovaries, they were divided into 502 LCL3-cows and 782 RCL3-cows (cows with an active CL on the left [L] or right [R] ovary, respectively). To induce estrus synchronization and investigate the effects of PGF2α administration on the incidence of estrus in both LCL3-cows and RCL3-cows, the cows were treated with one luteolytic dose of PGF2α and were inseminated after observed estrus (via visual observation lasting at least 30 minutes three times a day). To investigate the effects of side of ovulation at the time of PGF2α administration on reproductive parameters, pregnancy diagnosis was performed 28 days after insemination (using ultrasound) and 42 days after insemination (using transrectal palpation). The results showed that the percentage of the RCL3-cows was greater than the LCL3-cows (60.9% vs. 39.1%, respectively). Furthermore, ovulations switching from the left to right ovary in two successive ovulations were greater than those that switched from the right to left ovary. On the other hand, the sex ratio (male percentage) in the right uterine horn was greater than that of the left one. In the second experiment (in vitro), the developmental potential of bovine oocytes derived from the left (L-oocytes) and right (R-oocytes) ovaries after in vitro embryo production and heterogeneity in the developmental competence of L-oocytes and R-oocytes using the brilliant cresyl blue staining test as a selection criterion were evaluated. Results of the in vitro experiment showed that the percentage of cleavage and blastocyst rate of R-oocytes were greater (P < 0.001) than those of L-oocytes. Moreover, it appears that the side of ovaries had greater effects on the developmental competence of oocytes than other factors associated with heterogeneity in the developmental competence of oocytes, which can be detected by the brilliant cresyl blue test. In conclusion, the results of the in vivo study confirmed the observations in previous studies in which the right ovarian response (distribution of ovulation) was superior to that of the left ones. Interestingly, the in vitro experiments for the first time clearly showed that more ovulation on the right side is not the only reason for this unequal activity. In fact, in cattle, the greater developmental potential of oocytes originating from right ovaries may cause superior activity of the right side, and the effect is even higher than the differences in ovulation response between the left and right ovaries. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro differentiation of quail neural crest cells into sensory-like neuroblasts

NASA Technical Reports Server (NTRS)

Sieber-Blum, Maya; Kumar, Sanjiv R.; Riley, Danny A.

1988-01-01

Data are presented that demonstrate the ability of quail neural-crest embrionic cells grown as primary culture to differentiate in vitro into sensorylike neuroblasts. After 7-14 days of growth as primary culture, many of the putative sensory neuroblasts displayed substance P (SP)-like immunoreactivity and some exhibited histochemical carbonic anhydrase activity. Double staining experiments showed that the SP-like immunoreactive neuroblasts did not contain detectable levels of tyrosine hydroxylase or dopamine-beta-hydroxylase. The neuronal nature of the cultured sensorylike neuroblasts was further documented by double labeling for antibodies against the 68 kDa neurofilament polypeptide and substance P.

Preparation, characterization and bioactivities of Athelia rolfsii exopolysaccharide-zinc complex (AEPS-zinc).

PubMed

Dong, Jinman; Li, Hongmei; Min, Weihong

2018-07-01

A new Athelia rolfsii exopolysaccharides (AEPS) were purified by Sephacryl S-300 and S-200. The physicochemical characteristics of AEPS fractions were assayed by HPGPC and GC methods. The structures of AEPS and AEPS‑zinc complex were characterized by SEM, FTIR and NMR. Moreover, the bioactivities of complex were also evaluated by experiments in vitro and in vivo. AEPSI consisted of glucose, galacturonic acid, talose, galactose, mannose and xylose, the relative contents of them were 24.74, 19.60, 33.65, 8.77, 7.97 and 5.28%, respectively. AEPSII consisted of glucose, inositol, galacturonic acid, ribitol, gluconic acid, talose and xylose, whose relative contents were 36.06, 21.21, 12.78, 11.07, 6.58, 5.45 and 6.82%, respectively. The Mw and Mn of AEPSI were 6.1324×10 4 and 1.4218×10 4 Da, those of AEPSII were 517 and 248Da. SEM observations showed that microstructures of AEPS and AEPS‑zinc complex were obviously different both in size and shape. FTIR and NMR analysis indicated that AEPS might chelate with zinc ion through hydroxy and carboxy group. In vitro experiments showed that AEPS‑zinc complex had a good bioavailability, in vivo experiments showed that it had good effect on improving zinc deficiency and antioxidant activities, which suggested that it could be used as zinc supplementation with high antioxidant activities. Copyright © 2018 Elsevier B.V. All rights reserved.

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

PubMed

Hiroishi, S; Sugie, K; Yoshida, T; Morimoto, J; Taniguchi, Y; Imai, S; Kurebayashi, J

2001-06-26

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

Endogenous boldenone-formation in cattle: alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed.

PubMed

Verheyden, K; Noppe, H; Zorn, H; Van Immerseel, F; Vanden Bussche, J; Wille, K; Bekaert, K; Janssen, C R; De Brabander, H F; Vanhaecke, L

2010-04-01

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature. 2010 Elsevier Ltd. All rights reserved.

In vitro assessment of six aspiration catheters using a distal protection filter.

PubMed

Fujimura, Tatsuhiro; Okamura, Takayuki; Ando, Miyuki; Uchida, Kousuke; Tone, Takashi; Yonezawa, Fumio; Yano, Masafumi

2016-01-01

We assessed performance of 6 aspiration catheters for distal embolization using a distal protection filter in an in vitro experiment. In acute myocardial infarction, a distal protection filter is used for lesions likely to induce a distal embolism. Which aspiration cathether is most effective when used with a distal protection filter remains still unclear. A 0.5-cm3 bolus of gelatin as a model of stagnant pools of coronary plaque debris was captured in the distal protection filter and aspirated by 6 aspiration catheters. We measured and compared the length of the suspended embolus matter. Among the 6 catheters evaluated, the use of the Export Advance catheter (Medtronic) resulted in significantly shorter lengths of the suspended embolus matter compared to the use of the TVAC II (Nipro), Thrombuster III SL (Kaneka), and Rebirth Pro (Goodman) catheters (p < 0.01). The residual embolus matter in all cases had drained distally to the distal protection filter when the filter was retrieved. The use of the Export Advance catheter showed better performance using a distal protection filter in this in vitro experiment, and its use might be more effective in preventing distal embolisms in combination with a distal protection filter.

In-vitro and clinical evaluation of transurethral laser-induced prostatectomy (TULIP)

NASA Astrophysics Data System (ADS)

van Swol, Christiaan F. P.; Verdaasdonck, Rudolf M.; Mooibroek, Jaap; Boon, Tom A.

1993-05-01

Transurethral ultrasound-guided laser induced prostatectomy (TULIP) is a recent development in the treatment of benign prostatic hyperplasia. The system is based upon Nd:YAG laser irradiation delivered by a right angled fiber. The dosimetry used in a clinical situation is mostly based upon animal studies. In this study, the light and temperature distribution in the prostate during Nd:YAG laser irradiation were modeled using Monte Carlo and finite differences theory. The results of this model were compared with in vitro experiments. The influence of the different parameters involved, e.g., the scanning speed and the power of the laser beam, were evaluated. Initial results show the temperature distribution and thus the therapeutic effect of the TULIP procedure. Until now 36 patients have been treated successfully. The mean in-hospital time was somewhat shorter than for a TURP treatment while the results were comparable. These treatments, however, show the need for a better understanding of the mechanisms involved. Modeling and subsequent in vitro and in vivo measurements might improve the understanding and safe and successful application of prostate treatment using laser based systems.

In Vitro Biodegradation of Hepatotoxic Indospicine in Indigofera spicata and Its Degradation Derivatives by Camel Foregut and Cattle Rumen Fluids.

PubMed

Tan, Eddie T T; Al Jassim, Rafat; D'Arcy, Bruce R; Fletcher, Mary T

2017-08-30

The known accumulation of the hepatotoxin indospicine in tissues of camels and cattle grazing Indigofera pasture plants is unusual in that free amino acids would normally be expected to be degraded during the fermentation processes in these foregut fermenters. In this study, in vitro experiments were carried out to examine the degradability of indospicine of Indigofera spicata by camel and cattle foregut microbiota. In the first experiment, a 48 h in vitro incubation was carried out using foregut fluid samples that were collected from 15 feral camels and also a fistulated cow. Degradability of indospicine ranged between 97% and 99%, with the higher value of 99% for camels. A pooled sample of foregut fluids from three camels that were on a roughage diet was used in a second experiment to examine the time-dependent degradation of indospicine present in the plant materials. Results indicated that camels' foregut fluids have the ability to biodegrade ∼99% of the indospicine in I. spicata within 48 h of incubation and produced 2-aminopimelamic acid and 2-aminopimelic acid. The time-dependent degradation analysis showed rapid indospicine degradation (65 nmol/h) during the first 8-18 h of incubation followed by a slower degradation rate (12 nmol/h) between 18 and 48 h. Indospicine degradation products were also degraded toward the end of the experiment. The results of these in vitro degradation studies suggest that dietary indospicine may undergo extensive degradation in the foregut of the camel, resulting in trace levels after 48 h. The retention time for plant material in the camel foregut varies depending on feed quality, and the results of this study together with the observed accumulation of indospicine in camel tissues suggest that, although indospicine can be degraded by foregut fermentation, this degradation is not complete before the passage of the digesta into the intestine.

Evaluation of hair growth promoting activity of Phyllanthus niruri

PubMed Central

Patel, Satish; Sharma, Vikas; S. Chauhan, Nagendra; Thakur, Mayank; Dixit, Vinod Kumar

2015-01-01

Objective: This study was designed to investigate the potential Phyllanthus niruri (P. niruri ) extracts in promotion of hair growth. Materials and Methods: Here, we studied the hair growth promoting activity of petroleum ether extract of P. niruri following its topical administration. Alopecia was induced in albino rats by subcutaneous administration of testosterone for 21 days. Evaluation of hair loss inhibition was done by concurrent administration of extract and monitoring parameters like follicular density, anagen/telogen (A/T) ratio and histological observation of animal skin sections. Finasteride solution was applied topically as standard. In vitro experiments were also performed to study the effect of extract on the activity of 5α-reductase enzyme Results: Groups treated with petroleum ether extract of plant showed hair re-growth as reflected by follicular density, A/T ratio and skin sections. Histopathology and morphologic observations of hair re-growth at shaved sites showed active follicular proliferation. In vitro experiments results showed inhibitory activity of petroleum ether extract on type-2 5α-reductase enzyme and an increase in the amount of testosterone with increasing concentrations. Conclusion: It could be concluded that petroleum ether extracts of P. niruri might be useful in the treatment of testosterone-induced alopecia in the experimental animal by inhibiting 5α-reductase enzyme. PMID:26693408

Structure-function relationship of avian eggshell matrix proteins: a comparative study of two major eggshell matrix proteins, ansocalcin and OC-17.

PubMed

Lakshminarayanan, Rajamani; Joseph, Jeremiah S; Kini, R Manjunatha; Valiyaveettil, Suresh

2005-01-01

The role of individual matrix proteins in avian eggshell calcification is poorly understood despite numerous attempts to characterize and localize their presence in the eggshell matrix. Ansocalcin, the major matrix protein from goose eggshell, was found to induce the formation of calcite crystal aggregates under in vitro. Owing to its high similarity with the chicken eggshell matrix protein ovocleidin 17 (OC-17), a comparative investigation has been carried out to understand the structure-function relationship. RP-HPLC shows that ansocalcin is the major component in extracts of goose eggshells before and after bleach treatment. However, OC-17 was observed in minute quantities in the extract of bleach-treated chicken eggshells. In vitro crystal growth experiments showed that OC-17 and ansocalcin interact differently with the calcite crystals formed. Circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering studies showed that, under the conditions used in our experiments, OC-17 does not aggregate in solution or induce the nucleation of calcite aggregates in the concentration range used. These observations indicate that OC-17 and ansocalcin play different roles in the eggshell calcification. To our knowledge, this is the first report on the comparison of properties of homologous eggshell proteins that belong to the same phylogeny.

Synthesis and in vitro transfection efficiency of spermine-based cationic lipids with different central core structures and lipophilic tails.

PubMed

Niyomtham, Nattisa; Apiratikul, Nuttapon; Suksen, Kanoknetr; Opanasopit, Praneet; Yingyongnarongkul, Boon-Ek

2015-02-01

Twelve spermine-based cationic lipids with four different central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) and three hydrophobic tails (lauric acid, myristic acid and palmitic acid) were synthesized. The liposomes containing lipids and DOPE showed moderate to good in vitro DNA delivery into HeLa cells. GFP expression experiments revealed that liposomes composed of lipids with 3-amino-1,2-dioxypropyl as a central core structure exhibited highest transfection efficiency under serum-free condition. Whereas, lipid with 2-amino-1,3-dioxypropyl core structure showed highest transfection under 10% serum condition. Moreover, the liposomes and lipoplexes composted of these cationic lipids exhibited low cytotoxicity. Copyright © 2015. Published by Elsevier Ltd.

Novel inhibitors to Taenia solium Cu/Zn superoxide dismutase identified by virtual screening

NASA Astrophysics Data System (ADS)

García-Gutiérrez, P.; Landa-Piedra, A.; Rodríguez-Romero, A.; Parra-Unda, R.; Rojo-Domínguez, A.

2011-12-01

We describe in this work a successful virtual screening and experimental testing aimed to the identification of novel inhibitors of superoxide dismutase of the worm Taenia solium ( TsCu/Zn-SOD), a human parasite. Conformers from LeadQuest® database of drug-like compounds were selected and then docked on the surface of TsCu/Zn-SOD. Results were screened looking for ligand contacts with receptor side-chains not conserved in the human homologue, with a subsequent development of a score optimization by a set of energy minimization steps, aimed to identify lead compounds for in vitro experiments. Six out of fifty experimentally tested compounds showed μM inhibitory activity toward TsCu/Zn-SOD. Two of them showed species selectivity since did not inhibit the homologous human enzyme when assayed in vitro.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

PubMed

Gamrekelashvili, Jaba; Giagnorio, Roberto; Jussofie, Jasmin; Soehnlein, Oliver; Duchene, Johan; Briseño, Carlos G; Ramasamy, Saravana K; Krishnasamy, Kashyap; Limbourg, Anne; Kapanadze, Tamar; Ishifune, Chieko; Hinkel, Rabea; Radtke, Freddy; Strobl, Lothar J; Zimber-Strobl, Ursula; Napp, L Christian; Bauersachs, Johann; Haller, Hermann; Yasutomo, Koji; Kupatt, Christian; Murphy, Kenneth M; Adams, Ralf H; Weber, Christian; Limbourg, Florian P

2016-08-31

A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

PubMed Central

Gamrekelashvili, Jaba; Giagnorio, Roberto; Jussofie, Jasmin; Soehnlein, Oliver; Duchene, Johan; Briseño, Carlos G.; Ramasamy, Saravana K.; Krishnasamy, Kashyap; Limbourg, Anne; Häger, Christine; Kapanadze, Tamar; Ishifune, Chieko; Hinkel, Rabea; Radtke, Freddy; Strobl, Lothar J.; Zimber-Strobl, Ursula; Napp, L. Christian; Bauersachs, Johann; Haller, Hermann; Yasutomo, Koji; Kupatt, Christian; Murphy, Kenneth M.; Adams, Ralf H.; Weber, Christian; Limbourg, Florian P.

2016-01-01

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation. PMID:27576369

Downscaling the in vitro test of fungal bioremediation of polycyclic aromatic hydrocarbons: methodological approach.

PubMed

Drevinskas, Tomas; Mickienė, Rūta; Maruška, Audrius; Stankevičius, Mantas; Tiso, Nicola; Mikašauskaitė, Jurgita; Ragažinskienė, Ona; Levišauskas, Donatas; Bartkuvienė, Violeta; Snieškienė, Vilija; Stankevičienė, Antanina; Polcaro, Chiara; Galli, Emanuela; Donati, Enrica; Tekorius, Tomas; Kornyšova, Olga; Kaškonienė, Vilma

2016-02-01

The miniaturization and optimization of a white rot fungal bioremediation experiment is described in this paper. The optimized procedure allows determination of the degradation kinetics of anthracene. The miniaturized procedure requires only 2.5 ml of culture medium. The experiment is more precise, robust, and better controlled comparing it to classical tests in flasks. Using this technique, different parts, i.e., the culture medium, the fungi, and the cotton seal, can be analyzed. A simple sample preparation speeds up the analytical process. Experiments performed show degradation of anthracene up to approximately 60% by Irpex lacteus and up to approximately 40% by Pleurotus ostreatus in 25 days. Bioremediation of anthracene by the consortium of I. lacteus and P. ostreatus shows the biodegradation of anthracene up to approximately 56% in 23 days. At the end of the experiment, the surface tension of culture medium decreased comparing it to the blank, indicating generation of surfactant compounds.

Photobiomodulation of wound healing via visible and infrared laser irradiation.

PubMed

Solmaz, Hakan; Ulgen, Yekta; Gulsoy, Murat

2017-05-01

Fibroblast cells are known to be one of the key elements in wound healing process, which has been under the scope of research for decades. However, the exact mechanism of photobiomodulation on wound healing is not fully understood yet. Photobiomodulation of 635 and 809 nm laser irradiation at two different energy densities were investigated with two independent experiments; first, in vitro cell proliferation and then in vivo wound healing. L929 mouse fibroblast cell suspensions were exposed with 635 and 809 nm laser irradiations of 1 and 3 J/cm 2 energy densities at 50 mW output power separately for the investigation of photobiomodulation in vitro. Viabilities of cells were examined by means of MTT assays performed at the 24th, 48th, and 72nd hours following the laser irradiations. Following the in vitro experiments, 1 cm long cutaneous incisional skin wounds on Wistar albino rats (n = 24) were exposed with the same laser sources and doses in vivo. Wound samples were examined on 3rd, 5th, and 7th days of healing by means of mechanical tensile strength tests and histological examinations. MTT assay results showed that 635 nm laser irradiation of both energy densities after 24 h were found to be proliferative. One joule per square centimeter laser irradiation results also had positive effect on cell proliferation after 72 h. However, 809 nm laser irradiation at both energy densities had neither positive nor negative affects on cell viability. In vivo experiment results showed that, 635 nm laser irradiation of both energy densities stimulated wound healing in terms of tensile strength, whereas 809 nm laser stimulation did not cause any stimulative effect. The results of mechanical tests were compatible with the histological evaluations. In this study, it is observed that 635 nm laser irradiations of low energy densities had stimulative effects in terms of cell proliferation in vitro and mechanical strength of incisions in vivo. However, 809 nm laser irradiations at the same doses did not have any positive effect.

Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study

PubMed Central

2014-01-01

Endotoxins are part of the cell wall of Gram-negative bacteria. They are potent immune stimulators and can lead to death if present in high concentrations. Feed additives, which bind endotoxins in the gastrointestinal tract of animals, could help to prevent their negative impact. The objective of our study was to determine the potential of a bentonite (Bentonite 1), a sodium bentonite (Bentonite 2), a chemically treated smectite (Organoclay 1) and a modified attapulgite (Organoclay 2) to bind endotoxins in vitro. Polymyxin B served as positive control. The kinetic chromogenic Limulus Amebocyte lysate test was adapted to measure endotoxin activity. Firstly, a single sorption experiment (10 endotoxin units/mL (EU/mL)) was performed. Polymyxin B and organoclays showed 100% binding efficiency. Secondly, the adsorption efficiency of sorbents in aqueous solution with increasing endotoxin concentrations (2,450 – 51,700 EU/mL) was investigated. Organoclay 1 (0.1%) showed a good binding efficiency in aqueous solution (average 81%), whereas Bentonite 1 (0.1%) obtained a lower binding efficiency (21-54%). The following absorbent capacities were calculated in highest endotoxin concentration: 5.59 mg/g (Organoclay 1) > 3.97 mg/g (Polymyxin B) > 2.58mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Thirdly, a sorption experiment in artificial intestinal fluid was conducted. Especially for organoclays, which are known to be unspecific adsorbents, the endotoxin binding capacity was significantly reduced. In contrast, Bentonite 1 showed comparable results in artificial intestinal fluid and aqueous solution. Based on the results of this in vitro study, the effect of promising clay minerals will be investigated in in vivo trials. PMID:24383578

Effect of heat stress on the expression profile of Hsp90 among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breed of cattle: a comparative study.

PubMed

Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Kumar, Sushil; Singh, Rani; Sengar, G; Sharma, Arjava

2014-02-25

We evaluated the effect of thermal challenge on the expression profile of heat shock protein 90 (Hsp90) among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breeds of cattle. The present investigation was focused on the comparative studies on Hsp90 expression among Frieswal and Sahiwal under in vitro and environmental heat stress. Measured immediately after the in vitro heat shock to the peripheral blood mononuclear cells (PBMCs), the relative expression of Hsp90 mRNA was significantly (P<0.05) higher in Sahiwal compared to those in Frieswal. In later intervals of time, the differences in the expression levels between the two breeds become negligible coming down towards the basal level. A similar pattern was observed in the protein concentration showing significantly (P<0.05) higher levels in Sahiwal compared to those in Frieswal. The second sets of experiments were undertaken during summer months (March to May) when temperature peaked from 37 to 45 °C. During these months, Frieswal cows consistently recorded higher rectal temperatures than the Sahiwal breed. Further during this peak summer stress, Sahiwal showed significantly higher levels of mRNA transcripts as well as protein concentration compared to the Frieswal breed. Our findings also interestingly showed that, the cell viability of PBMC are significantly higher among the Sahiwal than Frieswal. Taken together, the experiments of both induced in vitro and environmental stress conditions indicate that, Sahiwal may express higher levels of Hsp90 then Frieswal to regulate their body temperature and increase cell survivality under heat stressed conditions. Copyright © 2013 Elsevier B.V. All rights reserved.

In vitro and in vivo studies on the transport of PEGylated silica nanoparticles across the blood-brain barrier.

PubMed

Liu, Dan; Lin, Bingqian; Shao, Wei; Zhu, Zhi; Ji, Tianhai; Yang, Chaoyong

2014-02-12

Transport of PEGylated silica nanoparticles (PSiNPs) with diameters of 100, 50, and 25 nm across the blood-brain barrier (BBB) was evaluated using an in vitro BBB model based on mouse cerebral endothelial cells (bEnd.3) cultured on transwell inserts within a chamber. In vivo animal experiments were further performed by noninvasive in vivo imaging and ex vivo optical imaging after injection via carotid artery. Confocal fluorescence studies were carried out to evaluate the uptake of PSiNPs by brain endothelial cells. The results showed that PSiNPs can traverse the BBB in vitro and in vivo. The transport efficiency of PSiNPs across BBB was found to be size-dependent, with increased particle size resulting in decreased efficiency. This work points to the potential application of small sized silica nanoparticles in brain imaging or drug delivery.

Antibacterial activities of amorphous cefuroxime axetil ultrafine particles prepared by high gravity antisolvent precipitation (HGAP).

PubMed

Zhao, Hong; Kang, Xu-liang; Chen, Xuan-li; Wang, Jie-xin; Le, Yuan; Shen, Zhi-gang; Chen, Jian-feng

2009-01-01

In vitro and in vivo antibacterial activities on the Staphylococcus aureus and Escherichia coli of the amorphous cefuroxime axetil (CFA) ultrafine particles prepared by HGAP method were investigated in this paper. The conventional sprayed CFA particles were studied as the control group. XRD, SEM, BET tests were performed to investigate the morphology changes of the samples before and after sterile. The in vitro dissolution test, minimal inhibitory concentrations (MIC) and the in vivo experiment on mice were explored. The results demonstrated that: (i) The structure, morphology and amorphous form of the particles could be affected during steam sterile process; (ii) CFA particles with different morphologies showed varied antibacterial activities; and (iii) the in vitro and in vivo antibacterial activities of the ultrafine particles prepared by HGAP is markedly stronger than that of the conventional sprayed amorphous particles.

Experimental research on the in vitro antitumor effects of Crataegus sanguinea.

PubMed

Sun, Jianling; Gao, Guolan; Gao, YuLian; Xiong, LiJuan; Li, Xiaoying; Guo, Jihong; Zhang, Yueming

2013-09-01

Crataegus sanguinea is a wild plant, which has been widely grown in the north and south of the Tianshan mountains in Xinjiang. In order to explore their anti-cancer properties, edible wild plants from Xinjiang have been tested for their antitumor properties. We used Ames tests, mouse bone marrow polychromatic erythrocytes micronucleus tests, and tumor cells cultured in vitro to study the anti-mutagenic and anti-tumor effects of C. sanguinea extract. We have shown that C. sanguinea has anti-mutagenic effect, but no mutagenicity. Cell culture in vitro experiments show that there is no inhibition of growth or increase in cell death on normal mouse fibroblasts, but a stronger inhibition of cell growth and an increase in cell death of Hep-2 and MGC-803 tumor cells. The results of this study illustrate that C. sanguinea extract has both anti-mutagenic and anti-tumor effects.

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer.

PubMed

Hinrichs, K; Love, C C; Brinsko, S P; Choi, Y H; Varner, D D

2002-07-01

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.

Sulphur tracer experiments in laboratory animals using 34S-labelled yeast.

PubMed

Martínez-Sierra, J Giner; Moreno Sanz, F; Herrero Espílez, P; Marchante Gayón, J M; Rodríguez Fernández, J; García Alonso, J I

2013-03-01

We have evaluated the use of (34)S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the (34)S-labelled yeast to laboratory animals to follow the fate and distribution of (34)S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of (34)S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.

Development of Biodegradable Implants for Use in Maxillofacial Surgery.

DTIC Science & Technology

1981-10-28

PAGE(WhM D .m4m 20 Abstract - continued -to obtain the desired composite material properties. In vitro -- experiments with these materials showed that...the composites were capable of retaining sufficient strength and integrity, in a pseudo-physiological environment, throughout the period normally...VII. Mechanical Properties of Reinforcing Fibers .......... .23 VIII. Effect of Sterilization on High-viscosity Polylactide Composites

CCL2/CCL5 secreted by the stroma induce IL-6/PYK2 dependent chemoresistance in ovarian cancer.

PubMed

Pasquier, Jennifer; Gosset, Marie; Geyl, Caroline; Hoarau-Véchot, Jessica; Chevrot, Audrey; Pocard, Marc; Mirshahi, Massoud; Lis, Raphael; Rafii, Arash; Touboul, Cyril

2018-02-19

Minimal residual disease is the main issue of advanced ovarian cancer treatment. According to the literature and previous results, we hypothesized that Mesenchymal Stromal Cells (MSC) could support this minimal residual disease by protecting ovarian cancer cells (OCC) from chemotherapy. In vitro study confirmed that MSC could induce OCC chemoresistance without contact using transwell setting. Further experiments showed that this induced chemoresistance was dependent on IL-6 OCC stimulation. We combined meticulous in vitro profiling and tumor xenograft models to study the role of IL-6 in MSC/OCC intereactions. We demonstrated that Tocilizumab® (anti-IL-6R therapy) in association with chemotherapy significantly reduced the peritoneal carcinosis index (PCI) than chemotherapy alone in mice xenografted with OCCs+MSCs. Further experiments showed that CCL2 and CCL5 are released by MSC in transwell co-culture and induce OCCs IL-6 secretion and chemoresistance. Finally, we found that IL-6 induced chemoresistance was dependent on PYK2 phosphorylation. These findings highlight the potential key role of the stroma in protecting minimal residual disease from chemotherapy, thus favoring recurrences. Future clinical trials targeting stroma could use anti-IL-6 therapy in association with chemotherapy.

Development of octreotide-conjugated polymeric prodrug of bufalin for targeted delivery to somatostatin receptor 2 overexpressing breast cancer in vitro and in vivo

PubMed Central

Liu, Tao; Jia, Tingting; Yuan, Xia; Liu, Cheng; Sun, Jian; Ni, Zhenhua; Xu, Jian; Wang, Xuhui; Yuan, Yi

2016-01-01

Background Development of polymeric prodrugs of small molecular anticancer drugs has become one of the most promising strategies to overcome the intrinsic shortcomings of small molecular anticancer drugs and improve their anticancer performance. Materials and methods In the current work, we fabricated a novel octreotide (Oct)-modified esterase-sensitive tumor-targeting polymeric prodrug of bufalin (BUF) and explored its anticancer performance against somatostatin receptor 2 overexpressing breast cancer. Results The obtained tumor-targeting polymeric prodrug of BUF, P(oligo[ethylene glycol] monomethyl ether methacrylate [OEGMA]-co-BUF-co-Oct), showed a nanosize dimension and controlled drug release features in the presence of esterase. It was demonstrated by in vitro experiment that P(OEGMA-co-BUF-co-Oct) showed enhanced cytotoxicity, cellular uptake, and apoptosis in comparison with those of free BUF. In vivo experiment further revealed the improved accumulation of drugs in tumor tissues and enhanced anticancer performance of P(OEGMA-co-BUF-co-Oct). Conclusion Taken together, this study indicated that polymeric prodrug of BUF holds promising potential toward the treatment of somatostatin receptor 2 overexpressing breast cancer. PMID:27284243

Effects of salicylic acid on Aspergillus flavus infection and aflatoxin B₁ accumulation in pistachio (Pistacia vera L.) fruit.

PubMed

Panahirad, Sima; Zaare-Nahandi, Fariborz; Mohammadi, Nilufar; Alizadeh-Salteh, Saeedeh; Safaie, Naser

2014-07-01

One of the most important saprophytic infections in fresh pistachio fruits after harvesting is Aspergillus flavus colonization, which significantly reduces fruit quality. Salicylic acid plays a crucial role in plant tissues and has a suppression effect on some fungi. The inhibitory effect of salicylic acid on the growth of A. flavus was assessed in vitro and in vivo. For this purpose, seven concentrations (0, 1, 3, 5, 7, 9 and 11 mmol L(-1)) of salicylic acid were used in both experiments. Also, aflatoxin B1 contents of the samples were analysed using immunoaffinity chromatography. The results obtained from in vitro experiments showed that salicylic acid significantly reduced Aspergillus growth at all concentrations, and at 9 mmol L(-1) growth was completely suppressed. In vivo evaluation showed relatively high levels of inhibition, though the intact treated fruits as compared with the injured treated fruits demonstrated higher inhibitory effects. Regarding the inhibitory effects of salicylic acid on the control of A. flavus contamination, its application on pistachio fruits after harvesting could be a promising approach to control the fungus infection and reduce aflatoxin production in treated fruits. © 2013 Society of Chemical Industry.

Prohibitin-mediated mitochondrial ubiquitination during spermiogenesis in Chinese mitten crab Eriocheir sinensis

PubMed Central

Hou, Cong-Cong; Wei, Chao-Guang; Lu, Cheng-Peng; Gao, Xin-Ming; Yang, Wan-Xi; Zhu, Jun-Quan

2017-01-01

The sperm of Eriocheir sinensis has a cup-shaped nucleus that contains several mitochondria embedded at the opening of the cup. The acrosome vesicle also contains derivants of mitochondria. The mitochondria distribution pattern involves a decrease in the number and changes in the structure and transportation of these organelles. The decreased number of sperm mitochondria is achieved through autophagy or the ubiquitination pathway. Prohibitin (PHB), the mitochondria inner membrane protein, is an evolutionarily highly conserved protein, is closely associated with spermatogenesis and sperm quality control and is also a potential substrate of ubiquitination. However, whether PHB protein mediates the ubiquitination pathway of sperm mitochondria in crustacean animals remains poorly understood. In the present study, we revealed that PHB, a substrate of ubiquitin, participates in the ubiquitination and degradation of mitochondria during spermiogenesis in E. sinensis. To confirm this finding, we used shRNA interference to reduce PHB expression and an overexpression technique to increase PHB expression in vitro. The interference experiment showed that the reduced PHB expression directly affected the polyubiquitination level and mitochondria status, whereas PHB overexpression markedly increased the polyubiquitination level. In vitro experiments also showed that PHB and its ubiquitination decide the fate of mitochondria. PMID:29228727

Research on oral microbiota of monozygotic twins with discordant caries experience - in vitro and in vivo study.

PubMed

Wu, Hongle; Zeng, Benhua; Li, Bolei; Ren, Biao; Zhao, Jianhua; Li, Mingyun; Peng, Xian; Feng, Mingye; Li, Jiyao; Wei, Hong; Cheng, Lei; Zhou, Xuedong

2018-05-08

Oral microbiome is potentially correlated with many diseases, such as dental caries, periodontitis, oral cancer and some systemic diseases. Twin model, as an effective method for studying human microbiota, is widely used in research of relationship between oral microbiota and dental caries. However, there were few researches focusing on caries discordant twins. In this study, in vitro assays were conducted combined with 16S rRNA sequencing analysis on oral microbiota sampled from twins who presented discordant caries experience and mice model was developed as well. Results showed that oral microbiota from caries-active twin possessed higher metabolic activity and produced more lactic production. 16S rRNA sequencing analysis showed that more than 80% of family taxa could be transferred into gnotobiotic-mice. Key caries-associated genera were significantly different between twins and the same difference in genus level could be found in mice as well (p < 0.05). This study suggested that oral microbiota of twins could be distinguished from each other despite the similarities in genetic make-up, living environment, and lifestyle. The difference in microbiota was applied to develop a mice model which may facilitate the investigation of core microbiota of dental caries.

Synthesis, structure, theoretical and experimental in vitro antioxidant/pharmacological properties of α-aryl, N-alkyl nitrones, as potential agents for the treatment of cerebral ischemia.

PubMed

Samadi, Abdelouahid; Soriano, Elena; Revuelta, Julia; Valderas, Carolina; Chioua, Mourad; Garrido, Ignacio; Bartolomé, Begoña; Tomassolli, Isabelle; Ismaili, Lhassane; González-Lafuente, Laura; Villarroya, Mercedes; García, Antonio G; Oset-Gasque, María J; Marco-Contelles, José

2011-01-15

The synthesis, structure, theoretical and experimental in vitro antioxidant properties using the DPPH, ORAC, and benzoic acid, as well as preliminary in vitro pharmacological activities of (Z)-α-aryl and heteroaryl N-alkyl-nitrones 6-15, 18, 19, 21, and 23, is reported. In the in vitro antioxidant activity, for the DPPH radical test, only nitrones bearing free phenol groups gave the best RSA (%) values, nitrones 13 and 14 showing the highest values in this assay. In the ORAC analysis, the most potent radical scavenger was nitrone indole 21, followed by the N-benzyl benzene-type nitrones 10 and 15. Interestingly enough, the archetypal nitrone 7 (PBN) gave a low RSA value (1.4%) in the DPPH test, or was inactive in the ORAC assay. Concerning the ability to scavenge the hydroxyl radical, all the nitrones studied proved active in this experiment, showing high values in the 94-97% range, the most potent being nitrone 14. The theoretical calculations for the prediction of the antioxidant power, and the potential of ionization confirm that nitrones 9 and 10 are among the best compounds in electron transfer processes, a result that is also in good agreement with the experimental values in the DPPH assay. The calculated energy values for the reaction of ROS (hydroxyl, peroxyl) with the nitrones predict that the most favourable adduct-spin will take place between nitrones 9, 10, and 21, a fact that would be in agreement with their experimentally observed scavenger ability. The in vitro pharmacological analysis showed that the neuroprotective profile of the target molecules was in general low, with values ranging from 0% to 18.7%, in human neuroblastoma cells stressed with a mixture of rotenone/oligomycin-A, being nitrones 18, and 6-8 the most potent, as they show values in the range 24-18.4%. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

The in vivo antitumor effects of type I-interferon against hepatocellular carcinoma: the suppression of tumor cell growth and angiogenesis.

PubMed

Enomoto, Hirayuki; Tao, Lihua; Eguchi, Ryoji; Sato, Ayuko; Honda, Masao; Kaneko, Shuichi; Iwata, Yoshinori; Nishikawa, Hiroki; Imanishi, Hiroyasu; Iijima, Hiroko; Tsujimura, Tohru; Nishiguchi, Shuhei

2017-09-22

Type I-interferon (IFN) is considered to exert antitumor effects through the inhibition of cancer cell proliferation and angiogenesis. Based on the species-specific biological activity of IFN, we evaluated each antitumor mechanism separately. We further examined the antitumor effects of type I-IFN combined with sorafenib. Human IFN (hIFN) significantly inhibited the proliferation of human hepatocellular carcinoma (HCC) Hep3B cells and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Although mouse IFN (mIFN) did not inhibit the proliferation of Hep3B cells in vitro, mIFN, as well as hIFN, showed significant antitumor effects in mouse Hep3B cell-xenograft model. Furthermore, mIFN treatment amplified the antitumor effects of sorafenib in vivo with the suppression of angiogenesis. The DNA chip analysis showed that the mIFN treatment promoted the antitumor signal pathways of sorafenib, including anti-angiogenic effects. Unlike the effects observed in in vitro experiments, mIFN showed an antitumor effect in the mouse Hep3B cell-xenograft model, suggesting a role of the anti-angiogenic activity in the in vivo tumoricidal effects of type I-IFN. In addition, our findings suggested the clinical utility of combination therapy with type І-IFN and sorafenib for HCC.

Resveratrol-cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

PubMed

Torres, V; Hamdi, M; Millán de la Blanca, M G; Urrego, R; Echeverri, J; López-Herrera, A; Rizos, D; Gutiérrez-Adán, A; Sánchez-Calabuig, M J

2018-03-26

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol-methyl β-cyclodextrin (RV-CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV-CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV-CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT-qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV-CD and embryo development and blastocysts gene expression by RT-qPCR were studied. A group without RV-CD (control - ) and a group with cyclodextrin (control + ) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p < .05). Blastocysts produced by IVC with resveratrol showed that RV-CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p < .05). RV-CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts. © 2018 Blackwell Verlag GmbH.

The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance.

PubMed

Scarth, James P; Spencer, Holly A; Timbers, Sarah E; Hudson, Simon C; Hillyer, Lynn L

2010-01-01

The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap.Using high resolution accurate mass full-scan analysis on the Orbitrap, the in vitro systems were found to generate at least the two most abundant phase I metabolites observed in vitro for all eight drugs studied. In the majority of cases, in vitro experiments were also able to generate the minor in vivo metabolites and sometimes metabolites that were only observed in vitro. More detailed analyses of fentanyl incubates using LC-MS/MS showed that it was possible to generate good quality spectra from the metabolites generated in vitro. These data support the suggestion of using in vitro incubates as metabolite reference material in place of in vivo post-administration samples in accordance with new qualitative identification guidelines in the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) document.In summary, the in vitro and in vivo phase I metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment, refine and reduce the existing equine in vivo paradigm. © 2010 John Wiley & Sons, Ltd.

Development of an integrated semi-automated system for in vitro pharmacodynamic modelling.

PubMed

Wang, Liangsu; Wismer, Michael K; Racine, Fred; Conway, Donald; Giacobbe, Robert A; Berejnaia, Olga; Kath, Gary S

2008-11-01

The aim of this study was to develop an integrated system for in vitro pharmacodynamic modelling of antimicrobials with greater flexibility, easier control and better accuracy than existing in vitro models. Custom-made bottle caps, fittings, valve controllers and a modified bench-top shaking incubator were used. A temperature-controlled automated sample collector was built. Computer software was developed to manage experiments and to control the entire system including solenoid pinch valves, peristaltic pumps and the sample collector. The system was validated by pharmacokinetic simulations of linezolid 600 mg infusion. The antibacterial effect of linezolid against multiple Staphylococcus aureus strains was also studied in this system. An integrated semi-automated bench-top system was built and validated. The temperature-controlled automated sample collector allowed unattended collection and temporary storage of samples. The system software reduced the labour necessary for many tasks and also improved the timing accuracy for performing simultaneous actions in multiple parallel experiments. The system was able to simulate human pharmacokinetics of linezolid 600 mg intravenous infusion accurately. A pharmacodynamic study of linezolid against multiple S. aureus strains with a range of MICs showed that the required 24 h free drug AUC/MIC ratio was approximately 30 in order to keep the organism counts at the same level as their initial inoculum and was about > or = 68 in order to achieve > 2 log(10) cfu/mL reduction in the in vitro model. The integrated semi-automated bench-top system provided the ability to overcome many of the drawbacks of existing in vitro models. It can be used for various simple or complicated pharmacokinetic/pharmacodynamic studies efficiently and conveniently.

Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase in vitro.

PubMed

McCue, Patrick P; Shetty, Kalidas

2004-01-01

Porcine pancreatic alpha-amylase (PPA) was allowed to react with herbal extracts containing rosmarinic acid (RA) and purified RA. The derivatized enzyme-phytochemical mixtures obtained were characterized for residual amylase activity. These in vitro experiments showed that the amylase activity was inhibited in the presence of these phytochemicals. The extent of amylase inhibition correlated with increased concentration of RA. RA-containing oregano extracts yielded higher than expected amylase inhibition than similar amount of purified RA, suggesting that other phenolic compounds or phenolic synergies may contribute to additional amylase inhibitory activity. The significance of food-grade, plant-based amylase inhibitors for modulation of diabetes mellitus and other oxidation-linked diseases is hypothesized and discussed.

A discrete in continuous mathematical model of cardiac progenitor cells formation and growth as spheroid clusters (Cardiospheres).

PubMed

Di Costanzo, Ezio; Giacomello, Alessandro; Messina, Elisa; Natalini, Roberto; Pontrelli, Giuseppe; Rossi, Fabrizio; Smits, Robert; Twarogowska, Monika

2018-03-14

We propose a discrete in continuous mathematical model describing the in vitro growth process of biophsy-derived mammalian cardiac progenitor cells growing as clusters in the form of spheres (Cardiospheres). The approach is hybrid: discrete at cellular scale and continuous at molecular level. In the present model, cells are subject to the self-organizing collective dynamics mechanism and, additionally, they can proliferate and differentiate, also depending on stochastic processes. The two latter processes are triggered and regulated by chemical signals present in the environment. Numerical simulations show the structure and the development of the clustered progenitors and are in a good agreement with the results obtained from in vitro experiments.

Visualization and Measurement of Flow in a Model Rotating-Wall Bioreactor

NASA Astrophysics Data System (ADS)

Brown, Jason B.; Neitzel, G. Paul

1997-11-01

Fluid shear has been observed to have an effect on the in vitro growth of mammalian cells and is expected to play a role in the in vitro development of aggregates of cells into tissue. The interactions between culture media and cell constructs within a circular Couette flow bioreactor with independently rotating cylinders are investigated in model studies using flow visualization. Particle-Image Velocimetry (PIV) is used to quantify the velocity field in a plane perpendicular to the vessel axis which contains a cell construct model. This velocity field is then used to compute the instantaneous shear field. Experiments show the path of the model cell construct is dependent on the rotation rates of the cylinders.

Photodynamic inactivation of microorganisms which cause pulmonary diseases with infrared light: an in vitro study

NASA Astrophysics Data System (ADS)

Leite, Ilaiáli S.; Geralde, Mariana C.; Salina, Ana C.; Medeiros, Alexandra I.; Kurachi, Cristina; Bagnato, Vanderlei S.; Inada, Natalia M.

2014-03-01

Lower respiratory infections are among the leading causes of death worldwide. In this study, it was evaluated the interaction of indocyanine green, a photosensitizer activated by infrared light, with alveolar macrophages and the effectiveness of the photodynamic therapy using this compound against Streptococcus pneumoniae . Initial experiments analyzed indocyanine green toxicity to alveolar macrophages in the dark with different drug concentrations and incubation times, and macrophage viability was obtained with the MTT method. The average of the results showed viability values below 90% for the two highest concentrations. Experiments with Streptococcus pneumoniae showed photodynamic inactivation with 10 μM indocyanine green solution. Further experiments with the bacteria in co-culture with AM will be conducted verifying the photodynamic inactivation effectiveness of the tested drug concentrations and incubation periods using infrared light.

Alginate/cashew gum floating bead as a matrix for larvicide release.

PubMed

Paula, Haroldo C B; de Oliveira, Erick F; Abreu, Flávia O M S; de Paula, Regina C M

2012-08-01

A polymeric floating system composed of Alginate (ALG) and Cashew gum (CG), loaded with an essential oil (Lippia sidoides-Ls) was prepared by ionotropic gelation, characterized regarding its physical-chemistry properties and evaluated on its potential as a controlled release system. The influence of process parameters on the buoyancy, loading, swelling and in vitro and in vivo release kinetics, was investigated. Results showed that beads produced with carbonate and Ls at high level contents exhibit good floatability (up to 5 days) and loading capacity (15.2-23.8%). In vitro release data showed a Fickian diffusion profile and in vivo experiments showed that ALG-CG floating system presented a superior and prolonged larvicide effect, in comparison with non-floating ones, presenting larvae mortality values of 85% and 33%, respectively, after 48 h. These results indicate that ALG-CG floating beads loaded with Ls presented enhanced oil entrapment efficiency, excellent floating ability, and suitable larvicide release pattern. Copyright © 2012 Elsevier B.V. All rights reserved.

Seismomorphogenesis: a novel approach to acclimatization of tissue culture regenerated plants.

PubMed

Sarmast, Mostafa Khoshhal; Salehi, Hassan; Khosh-Khui, Morteza

2014-12-01

Plantlets under in vitro conditions transferred to ex vivo conditions are exposed to biotic and abiotic stresses. Furthermore, in vitro regenerated plants are typically frail and sometimes difficult to handle subsequently increasing their risk to damage and disease; hence acclimatization of these plantlets is the most important step in tissue culture techniques. An experiment was conducted under in vitro conditions to study the effects of shaking duration (twice daily at 6:00 a.m. and 9:00 p.m. for 2, 4, 8, and 16 min at 250 rpm for 14 days) on Sansevieria trifasciata L. as a model plant. Results showed that shaking improved handling, total plant height, and leaf characteristics of the model plant. Forty-eight hours after 14 days of shaking treatments with increasing shaking time, leaf length decreased but proline content of leaf increased. However, 6 months after starting the experiment different results were observed. In explants that received 16 min of shaking treatment, leaf length and area and photosynthesis rate were increased compared with control plantlets. Six months after starting the experiment, control plantlets had 12.5 % mortality; however, no mortality was observed in other treated explants. The results demonstrated that shaking improved the explants' root length and number and as a simple, cost-effective, and non-chemical novel approach may be substituted for other prevalent acclimatization techniques used for tissue culture regenerated plantlets. Further studies with sensitive plants are needed to establish this hypothesis.

Ex vivo blood vessel bioreactor for analysis of the biodegradation of magnesium stent models with and without vessel wall integration.

PubMed

Wang, Juan; Liu, Lumei; Wu, Yifan; Maitz, Manfred F; Wang, Zhihong; Koo, Youngmi; Zhao, Ansha; Sankar, Jagannathan; Kong, Deling; Huang, Nan; Yun, Yeoheung

2017-03-01

Current in vitro models fail in predicting the degradation rate and mode of magnesium (Mg) stents in vivo. To overcome this, the microenvironment of the stent is simulated here in an ex vivo bioreactor with porcine aorta and circulating medium, and compared with standard static in vitro immersion and with in vivo rat aorta models. In ex vivo and in vivo conditions, pure Mg wires were exposed to the aortic lumen and inserted into the aortic wall to mimic early- and long-term implantation, respectively. Results showed that: 1) Degradation rates of Mg were similar for all the fluid diffusion conditions (in vitro static, aortic wall ex vivo and in vivo); however, Mg degradation under flow condition (i.e. in the lumen) in vivo was slower than ex vivo; 2) The corrosion mode in the samples can be mainly described as localized (in vitro), mixed localized and uniform (ex vivo), and uniform (in vivo); 3) Abundant degradation products (MgO/Mg(OH) 2 and Ca/P) with gas bubbles accumulated around the localized degradation regions ex vivo, but a uniform and thin degradation product layer was found in vivo. It is concluded that the ex vivo vascular bioreactor provides an improved test setting for magnesium degradation between static immersion and animal experiments and highlights its promising role in bridging degradation behavior and biological response for vascular stent research. Magnesium and its alloys are candidates for a new generation of biodegradable stent materials. However, the in vitro degradation of magnesium stents does not match the clinical degradation rates, corrupting the validity of conventional degradation tests. Here we report an ex vivo vascular bioreactor, which allows simulation of the microenvironment with and without blood vessel integration to study the biodegradation of magnesium implants in comparison with standard in vitro test conditions and with in vivo implantations. The bioreactor did simulate the corrosion of an intramural implant very well, but showed too high degradation for non-covered implants. It is concluded that this system is in between static incubation and animal experiments concerning the predictivity of the degradation. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

An in vitro reprogrammable antiviral RISC with size-preferential ribonuclease activity.

PubMed

Omarov, Rustem T; Ciomperlik, Jessica; Scholthof, Herman B

2016-03-01

Infection of Nicotiana benthamiana plants with Tomato bushy stunt virus (TBSV) mutants compromised for silencing suppression induces formation of an antiviral RISC (vRISC) that can be isolated using chromatography procedures. The isolated vRISC sequence-specifically degrades TBSV RNA in vitro, its activity can be down-regulated by removing siRNAs, and re-stimulated by exogenous supply of siRNAs. vRISC is most effective at hydrolyzing the ~4.8kb genomic RNA, but less so for a ~2.2kb TBSV subgenomic mRNA (sgRNA1), while the 3' co-terminal sgRNA2 of ~0.9kb appears insensitive to vRISC cleavage. Moreover, experiments with in vitro generated 5' co-terminal viral transcripts show that RNAs of ~2.7kb are efficiently cleaved while those of ~1.1kb or shorter are unaffected. The isolated antiviral ribonuclease complex fails to degrade ~0.4kb defective interfering RNAs (DIs) in vitro, agreeing with findings that in plants DIs are not targeted by silencing. Copyright © 2016. Published by Elsevier Inc.

Comparison of colony stimulation factors on in vitro rat and human neutrophil function.

PubMed

Wheeler, J G; Huffine, M E; Childress, S; Sikes, J

1994-01-01

The effects of rhCSFs on in vitro polymorphonuclear leukocyte (PMN) function were studied in Sprague-Dawley neonatal and adult rats and adult and umbilical cord derived human PMN to compare species response. Following in vitro exposure to buffer or rhCSFs (50-100 micrograms/ml), PMN oxidative burst, chemotactic activity and adherence protein expression were measured. RhG-CSF increased the oxidative burst of adult rat PMN as measured by chemiluminescence and altered CD11b/CD18 in resting neonatal rat but not adult rat cells. RhGM-CSF had no effect on adult rat PMN function in vitro, but led to modest changes in adult rat PMN diapedesis across rat peritoneum. No responses were noted to rhM-CSF. Human PMN responded best to GM-CSF (particularly in the neonate), intermediately to G-CSF and none to M-CSF. These experiments show that the profile of cytokine effects is not similar in adult and neonatal rat PMN when compared to human cells. The diversity of actions in other species must be considered when using rhCSFs in animal models.

Design of experiments (DOE) - history, concepts, and relevance to in vitro culture

USDA-ARS?s Scientific Manuscript database

Design of experiments (DOE) is a large and well-developed field for understanding and improving the performance of complex systems. Because in vitro culture systems are complex, but easily manipulated in controlled conditions, they are particularly well-suited for the application of DOE principle...

In Vitro and In Silico Antidiabetic and Antimicrobial Evaluation of Constituents from Kickxia ramosissima (Nanorrhinum ramosissimum)

PubMed Central

Amin, Adnan; Tuenter, Emmy; Foubert, Kenn; Iqbal, Jamhsed; Cos, Paul; Maes, Louis; Exarchou, Vassiliki; Apers, Sandra; Pieters, Luc

2017-01-01

Background and Aims: Kickxia ramosissima (Wall.) Janch (or Nanorrhinum ramosissimum (Wall.) Betsche is a well-known medicinal plant in Pakistan that is traditionally used in diabetic and inflammatory conditions. Because little information is available on its phytochemical composition, a range of constituents were isolated and evaluated in vitro in assays related to the traditional use. Methods: Dried whole plant material was extracted and chromatographically fractionated. Isolated constituents were evaluated in silico and in vitro in assays related to the traditional use against diabetes (inhibition of α-glucosidase activity; inhibition of advanced glycation endproducts) and in inflammatory conditions (inhibition of AAPH induced linoleic acid peroxidation, inhibition of 15-LOX, antimicrobial activity). Results: Phytochemical analysis of the extracts and fractions led to isolation of 7 compounds, including the iridoids kickxiasine (being a new compound), mussaenosidic acid, mussaenoside and linarioside; the flavonoids pectolinarigenin and pectolinarin; and 4-hydroxy-benzoic acid methyl ester. The iridoids showed weak antiglycation activity. The flavonoids, however, showed interesting results as pectolinarigenin was highly active compared to pectolinarin. In the α-glucosidase inhibition assay, only weak activity was observed for the iridoids. However, the flavonoid pectolinarigenin showed good activity, followed by pectolinarin. In the 15-LOX experiment, moderate inhibition was recorded for most compounds, the iridoids mussaenosidic acid and mussaenoside being the most active. In the AAPH assay, weak or no inhibition was recorded for all compounds. The in silico assays for the α-glucosidase and 15-LOX assays confirmed the results of respective in vitro assays. Pectolinarigenin showed moderate antimicrobial activity against Staphylococcus aureus, Plasmodium falciparum K1, and Trypanosoma cruzi, but it was not cytotoxic on a human MRC-5 cell line. Conclusion: Our findings may in part contribute to explain the traditional use of K. ramosissima. PMID:28507520

Ion Beam Sputtered Coatings of Bioglass

NASA Technical Reports Server (NTRS)

Hench, Larry L.; Wilson, J.; Ruzakowski, Patricia Henrietta Anne

1982-01-01

The ion beam sputtering technique available at the NASA-Lewis was used to apply coatings of bioglass to ceramic, metallic, and polymeric substrates. Experiments in vivo and in vitro described investigate these coatings. Some degree of substrate masking was obtained in all samples although stability and reactivity equivalent to bulk bioglass was not observed in all coated samples. Some degree of stability was seen in all coated samples that were reacted in vitro. Both metallic and ceramic substrates coated in this manner failed to show significantly improved coatings over those obtained with existing techniques. Implantation of the coated ceramic substrate samples in bone gave no definite bonding as seen with bulk glass; however, partial and patchy bonding was seen. Polymeric substrates in these studies showed promise of success. The coatings applied were sufficient to mask the underlying reactive test surface and tissue adhesion of collagen to bioglass was seen. Hydrophilic, hydrophobic, charged, and uncharged polymeric surfaces were successfully coated.

Radioprotective activity of Gentiana lutea extract and mangiferin.

PubMed

Menkovic, Nebojsa; Juranic, Zorica; Stanojkovic, Tatjana; Raonic-Stevanovic, Tatjana; Savikin, Katarina; Zdunić, Gordana; Borojevic, Nenad

2010-11-01

Radioprotective/sensitizing actions of Gentiana lutea aqueous-ethanol extract and mangiferin on radiation-induced effects on different types of cells were investigated. The study focused on the decreasing survival of normal human immunocompetent cells, the survival of the malignant cells in vitro, and the survival of ex vivo irradiated cells before and after consumption of the extract by healthy volunteers. The in vitro experiments showed that mangiferin could inhibit cytotoxic action of ionizing irradiation (doses of 6 and 8 Gy) only on normal resting human PBMC, not stimulated for proliferation. Orally consumed G. lutea extract showed the potential to reduce the cytotoxic effect of x-ray irradiation on normal human immunocompetent cells PBMC of some healthy people, without changing the susceptibility of malignant cells to be destroyed by irradiation. Since the radioprotective effect was individually dependent, further clinical studies are needed. Copyright © 2010 John Wiley & Sons, Ltd.

Evidence of oleuropein degradation by olive leaf protein extract.

PubMed

De Leonardis, Antonella; Macciola, Vincenzo; Cuomo, Francesca; Lopez, Francesco

2015-05-15

The enzymatic activity of raw protein olive leaf extract has been investigated in vivo, on olive leaf homogenate and, in vitro with pure oleuropein and other phenolic substrates. At least two types of enzymes were found to be involved in the degradation of endogenous oleuropein in olive leaves. As for the in vitro experiments, the presence of active polyphenoloxidase and β-glucosidase was determined by HPLC and UV-Visible spectroscopy. Interestingly, both the enzymatic activities were found to change during the storage of olive leaves. Specifically, the protein extracts obtained from fresh leaves showed the presence of both the enzymatic activities, because oleuropein depletion occurred simultaneously with the formation of the oleuropein aglycon, 3,4-DHPEA-EA. In comparison leaves subjected to the drying process showed a polyphenoloxidase activity leading exclusively to the formation of oxidation products responsible for the typical brown coloration of the reaction solution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reconstructing Ancient Forms of Life

NASA Technical Reports Server (NTRS)

Benner, Steven A.

1998-01-01

Progress in the past three months has occurred in two areas, reconstruction of ancestral proteins and improved understanding of chemical features that are likely to be universal in generic matter regardless of its genesis. Ancestral ribonucleases have been reconstructed, and an example has been developed that shows how physiological function can be assigned to in vitro behaviors observed in biological systems. Sequence data have been collected to permit the reconstruction of src homology 2 domains that underwent radiative divergence at the time of the radiative divergence of chordates. New studies have been completed that show how genetic matter (or its remnants) might be detected on Mars (or other non-terrean locations.) Last, the first in vitro selection experiments have been completed using a nucleoside library carrying positively charged functionality, illustrating the importance of non-standard nucleotides to those attempting to obtain evidence for an "RNA world" as an early episode of life on earth.

False HDAC Inhibition by Aurone Compound.

PubMed

Itoh, Yukihiro; Suzuki, Miki; Matsui, Taiji; Ota, Yosuke; Hui, Zi; Tsubaki, Kazunori; Suzuki, Takayoshi

2016-01-01

Fluorescence assays are useful tools for estimating enzymatic activity. Their simplicity and manageability make them suitable for screening enzyme inhibitors in drug discovery studies. However, researchers need to pay attention to compounds that show auto-fluorescence and quench fluorescence, because such compounds lower the accuracy of the fluorescence assay systems by producing false-positive or negative results. In this study, we found that aurone compound 7, which has been reported as a histone deacetylase (HDAC) inhibitor, gave false-positive results. Although compound 7 was identified by an in vitro HDAC fluorescence assay, it did not show HDAC inhibitory activity in a cell-based assay, leading us to suspect its in vitro HDAC inhibitory activity. As a result of verification experiments, we found that compound 7 interferes with the HDAC fluorescence assay by quenching the HDAC fluorescence signal. Our findings underscore the faults of fluorescence assays and call attention to careless interpretation.

Antisense oligonucleotides effectively inhibit the co-transcriptional splicing of a Candida group I intron in vitro and in vivo: Implications for antifungal therapeutics.

PubMed

Zhang, Libin; Leibowitz, Michael J; Zhang, Yi

2009-02-18

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3-P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

Comparative evaluation of rivastigmine permeation from a transdermal system in the Franz cell using synthetic membranes and pig ear skin with in vivo-in vitro correlation.

PubMed

Simon, Alice; Amaro, Maria Inês; Healy, Anne Marie; Cabral, Lucio Mendes; de Sousa, Valeria Pereira

2016-10-15

In the present study, in vitro permeation experiments in a Franz diffusion cell were performed using different synthetic polymeric membranes and pig ear skin to evaluate a rivastigmine (RV) transdermal drug delivery system. In vitro-in vivo correlations (IVIVC) were examined to determine the best model membrane. In vitro permeation studies across different synthetic membranes and skin were performed for the Exelon(®) Patch (which contains RV), and the results were compared. Deconvolution of bioavailability data using the Wagner-Nelson method enabled the fraction of RV absorbed to be determined and a point-to-point IVIVC to be established. The synthetic membrane, Strat-M™, showed a RV permeation profile similar to that obtained with pig ear skin (R(2)=0.920). Studies with Strat-M™ resulted in a good and linear IVIVC (R(2)=0.991) when compared with other synthetic membranes that showed R(2) values less than 0.90. The R(2) for pig ear skin was 0.982. Strat-M™ membrane was the only synthetic membrane that adequately simulated skin barrier performance and therefore it can be considered to be a suitable alternative to human or animal skin in evaluating transdermal drug transport, potentially reducing the number of studies requiring human or animal samples. Copyright © 2016 Elsevier B.V. All rights reserved.

In vitro and in vivo comparisons of constant resistance AC iontophoresis and DC iontophoresis.

PubMed

Li, S Kevin; Higuchi, William I; Zhu, Honggang; Kern, Steven E; Miller, David J; Hastings, Matthew S

2003-09-04

A previous in vitro constant electrical resistance alternating current (AC) iontophoresis study with human epidermal membrane (HEM) and a model neutral permeant has shown less inter- and intra-sample variability in iontophoretic transport relative to conventional constant direct current (DC) iontophoresis. The objectives of the present study were to address the following questions. (1) Can the skin electrical resistance be maintained at a constant level by AC in humans in vivo? (2) Are the in vitro data with HEM representative of those in vivo? (3) Does constant skin resistance AC iontophoresis have less inter- and intra-sample variability than conventional constant current DC iontophoresis in vivo? (4) What are the electrical and the barrier properties of skin during iontophoresis in vivo? In the present study, in vitro HEM experiments were carried out with the constant resistance AC and the conventional constant current DC methods using mannitol and glucose as the neutral model permeants. In vivo human experiments were performed using glucose as the permeant with a constant skin resistance AC only protocol and two conventional constant current DC methods (continuous constant current DC and constant current DC with its polarity alternated every 10 min with a 3:7 on:off duty cycle). Constant current DC iontophoresis was conducted with commercial constant current DC devices, and constant resistance AC iontophoresis was carried out by reducing and maintaining the skin resistance at a constant target value with AC supplied from a function generator. This study shows that (1) skin electrical resistance can be maintained at a constant level during AC iontophoresis in vivo; (2) HEM in vitro and human skin in vivo demonstrate similar electrical and barrier properties, and these properties are consistent with our previous findings; (3) there is general qualitative and semi-quantitative agreement between the HEM data in vitro and human skin data in vivo; and (4) constant skin resistance AC iontophoresis generally provides less inter- and intra-subject variability than conventional constant current DC.

In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

PubMed Central

Lepoittevin, Camille; Frigerio, Jean-Marc; Garnier-Géré, Pauline; Salin, Franck; Cervera, María-Teresa; Vornam, Barbara; Harvengt, Luc; Plomion, Christophe

2010-01-01

Background There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C). Methodology/Principal Findings A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). Conclusions/Significance This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome. PMID:20543950

Development of a statistical model for cervical cancer cell death with irreversible electroporation in vitro.

PubMed

Yang, Yongji; Moser, Michael A J; Zhang, Edwin; Zhang, Wenjun; Zhang, Bing

2018-01-01

The aim of this study was to develop a statistical model for cell death by irreversible electroporation (IRE) and to show that the statistic model is more accurate than the electric field threshold model in the literature using cervical cancer cells in vitro. HeLa cell line was cultured and treated with different IRE protocols in order to obtain data for modeling the statistical relationship between the cell death and pulse-setting parameters. In total, 340 in vitro experiments were performed with a commercial IRE pulse system, including a pulse generator and an electric cuvette. Trypan blue staining technique was used to evaluate cell death after 4 hours of incubation following IRE treatment. Peleg-Fermi model was used in the study to build the statistical relationship using the cell viability data obtained from the in vitro experiments. A finite element model of IRE for the electric field distribution was also built. Comparison of ablation zones between the statistical model and electric threshold model (drawn from the finite element model) was used to show the accuracy of the proposed statistical model in the description of the ablation zone and its applicability in different pulse-setting parameters. The statistical models describing the relationships between HeLa cell death and pulse length and the number of pulses, respectively, were built. The values of the curve fitting parameters were obtained using the Peleg-Fermi model for the treatment of cervical cancer with IRE. The difference in the ablation zone between the statistical model and the electric threshold model was also illustrated to show the accuracy of the proposed statistical model in the representation of ablation zone in IRE. This study concluded that: (1) the proposed statistical model accurately described the ablation zone of IRE with cervical cancer cells, and was more accurate compared with the electric field model; (2) the proposed statistical model was able to estimate the value of electric field threshold for the computer simulation of IRE in the treatment of cervical cancer; and (3) the proposed statistical model was able to express the change in ablation zone with the change in pulse-setting parameters.

Use of a nanoporous biodegradable miniature device to regulate cytokine release for cancer treatment

PubMed Central

He, Hongyan; Grignol, Valerie; Karpa, Volodymyr; Yen, Chi; LaPerle, Krista; Zhang, Xiaoli; Jones, Natalie B.; Liang, Margaret I.; Lesinski, Gregory B.; Ho, W.S. Winston; Carson, William E.; Lee, L. James

2014-01-01

The clinical management of locally recurrent or unresectable malignant melanoma continues to pose a significant challenge. These lesions are typically painful and currently available treatments, such as repeated intratumoral injections of interferon-alpha (IFN-α), are costly and inconvenient. Nanotechnology offers promise as a novel means of drug delivery. A capsule-like nanoporous miniature device (NMD) based on a biodegradable polymer, poly(polycaprolactone) (PCL) was developed for controlling the local delivery of immunological agents to the tumor microenvironment. The device consists of a nanoporous release gate, a fabricated drug reservoir loaded with IFN-α and a protective layer. To improve the biocompatibility of the device, a hydrophilic poly(ethylene glycol) monoacrylate was applied to the outside wall of the device via covalent bonding techniques. Microscopic visualization of the nanoporous gate from in vitro experiments exhibited good pore stability over a two-month period. In vitro experiments demonstrated a constant release rate of IFN-α from the NMD and showed that the release rate could be regulated by the gate area. The released IFN-α was biologically functional. Cytokine-containing supernatants from release experiments phosphorylated signal transducer and activator of transcription (STAT1) in peripheral blood mononuclear cells. Subcutaneous implantation of the NMDs was well tolerated and associated with an anti-tumor effect in a human xenograft model of melanoma. There was no evidence of a significant inflammatory response to the NMD or encapsulation of the NMD by fibrosis. These experiments show that the NMD can be fabricated and employed in vivo as a versatile drug delivery platform. PMID:21362447

Diffusion of Antimicrobials Across Silicone Hydrogel Contact Lenses.

PubMed

Zambelli, Alison M; Brothers, Kimberly M; Hunt, Kristin M; Romanowski, Eric G; Nau, Amy C; Dhaliwal, Deepinder K; Shanks, Robert M Q

2015-09-01

To measure the diffusion of topical preparations of moxifloxacin, amphotericin B (AmB), and polyhexamethylene biguanide (PHMB) through silicone hydrogel (SH) contact lenses (CLs) in vitro. Using an in vitro model, the diffusion of three antimicrobials through SH CLs was measured. Diffused compounds were measured using a spectrophotometer at set time points over a period of 4 hr. The amount of each diffused antimicrobial was determined by comparing the experimental value with a standard curve. A biological assay was performed to validate the CL diffusion assay by testing antimicrobial activity of diffused material against lawns of susceptible bacteria (Staphylococcus epidermidis) and yeast (Saccharomyces cerevisiae). Experiments were repeated at least two times with a total of at least four independent replicates. Our data show detectable moxifloxacin and PHMB diffusion through SH CLs at 30 min, whereas AmB diffusion remained below the limit of detection within the 4-hr experimental period. In the biological assay, diffused moxifloxacin demonstrated microbial killing starting at 20 min on bacterial lawns, whereas PHMB and AmB failed to demonstrate killing on microbial lawns over the course of the 60-min experiment. In vitro diffusion assays demonstrate limited penetration of certain anti-infective agents through SH CLs. Further studies regarding the clinical benefit of using these agents along with bandage CL for corneal pathologic condition are warranted.

Differences in response of two model estuarine crustaceans after lethal and sublethal exposures to chlorpyrifos.

PubMed

Key, Peter B; Simonik, Elizabeth; Kish, Nicole; Chung, Katy W; Fulton, Michael H

2013-01-01

This study assessed the in vitro and in vivo effects of an acetylcholinesterase enzyme inhibitor (chlorpyrifos) in two estuarine crustaceans: grass shrimp (Palaemonetes pugio) and mysid (Americamysis bahia). The differences in response were quantified after lethal and sublethal exposures to chlorpyrifos and in vitro assays with chlorpyrifos-oxon. Results from the in vitro experiments indicated that the target enzyme, acetylcholinesterase (AChE), in the two species was similar in sensitivity to chlorpyrifos inhibition with IC50s of 0.98 nM and 0.89 nM for grass shrimp and mysids, respectively. In vivo experiments showed that mysids were significantly more sensitive to chlorpyrifos-induced AChE inhibition after 24 h of exposure. The in vivo EC50s for AChE inhibition were 1.23 μg L(-1) for grass shrimp and 0.027 μg L(-1) for mysids. Median lethal concentrations (24h LC50 values) were 1.06 μg L(-1) for grass shrimp and 0.068 μg L(-1) for mysids. The results suggest that differences in the response of these two crustaceans are likely related to differences in uptake and metabolism rather than target site sensitivity.

In vitro transcriptomic prediction of hepatotoxicity for early drug discovery

PubMed Central

Cheng, Feng; Theodorescu, Dan; Schulman, Ira G.; Lee, Jae K.

2012-01-01

Liver toxicity (hepatotoxicity) is a critical issue in drug discovery and development. Standard preclinical evaluation of drug hepatotoxicity is generally performed using in vivo animal systems. However, only a small number of preselected compounds can be examined in vivo due to high experimental costs. A more efficient yet accurate screening technique which can identify potentially hepatotoxic compounds in the early stages of drug development would thus be valuable. Here, we develop and apply a novel genomic prediction technique for screening hepatotoxic compounds based on in vitro human liver cell tests. Using a training set of in vivo rodent experiments for drug hepatotoxicity evaluation, we discovered common biomarkers of drug-induced liver toxicity among six heterogeneous compounds. This gene set was further triaged to a subset of 32 genes that can be used as a multi-gene expression signature to predict hepatotoxicity. This multi-gene predictor was independently validated and showed consistently high prediction performance on five test sets of in vitro human liver cell and in vivo animal toxicity experiments. The predictor also demonstrated utility in evaluating different degrees of toxicity in response to drug concentrations which may be useful not only for discerning a compound’s general hepatotoxicity but also for determining its toxic concentration. PMID:21884709

Non-invasive transcranial ultrasound therapy based on a 3D CT scan: protocol validation and in vitro results

NASA Astrophysics Data System (ADS)

Marquet, F.; Pernot, M.; Aubry, J.-F.; Montaldo, G.; Marsac, L.; Tanter, M.; Fink, M.

2009-05-01

A non-invasive protocol for transcranial brain tissue ablation with ultrasound is studied and validated in vitro. The skull induces strong aberrations both in phase and in amplitude, resulting in a severe degradation of the beam shape. Adaptive corrections of the distortions induced by the skull bone are performed using a previous 3D computational tomography scan acquisition (CT) of the skull bone structure. These CT scan data are used as entry parameters in a FDTD (finite differences time domain) simulation of the full wave propagation equation. A numerical computation is used to deduce the impulse response relating the targeted location and the ultrasound therapeutic array, thus providing a virtual time-reversal mirror. This impulse response is then time-reversed and transmitted experimentally by a therapeutic array positioned exactly in the same referential frame as the one used during CT scan acquisitions. In vitro experiments are conducted on monkey and human skull specimens using an array of 300 transmit elements working at a central frequency of 1 MHz. These experiments show a precise refocusing of the ultrasonic beam at the targeted location with a positioning error lower than 0.7 mm. The complete validation of this transcranial adaptive focusing procedure paves the way to in vivo animal and human transcranial HIFU investigations.

Oleo gum resin of Ferula assa-foetida L. ameliorates peripheral neuropathy in mice.

PubMed

Homayouni Moghadam, Farshad; Dehghan, Maryam; Zarepur, Ehsan; Dehlavi, Reyhaneh; Ghaseminia, Fatemeh; Ehsani, Shima; Mohammadzadeh, Golnaz; Barzegar, Kazem

2014-05-28

According to the Chinese, European, Iranian and Indian traditional medicines, oleo gum resin of Ferula assa-foetida (asafoetida) has therapeutic effects on different kinds of diseases. Some of these effects are related to the diseases of nervous system such as hysteresis and convulsion. In recent studies, some anti-epileptic and neuroprotective roles were also considered for it and we examined its possible role on treatment of peripheral neuropathy. in vitro studies were carried out to identify the response of isolated sciatic nerves to different concentrations of oleo gum resin of asafoetida solved in Lock׳s solution. Then, in vivo studies were conducted to evaluate its effect on amelioration of peripheral neuropathy in mice. Peripheral neuropathy was induced by intraperiotoneal injection of high doses of pyridoxine in adult Balb/c male mice. Tail flick tests were performed to identify the incidence of neuropathy in animals. After 10 days treatment with asafoetida, the efficiency of treatment was assessed by behavioral, electrophysiological and histological studies. in vitro experiments confirmed that incubating the nerves in aqueous extract of oleo gum rein of asafoetida increased the amplitude and decreased the latent period of nerve compound action potential (CAP). Nerve conduction velocity (NCV) and amplitude of CAP also improved in asafoetida treated animals. Histological and behavioral studies showed that asafoetida was able to facilitate the healing process in peripheral nerves. in vitro experiments showed that asafoetida is a nerve stimulant and its administration in neuropathic mice exerted neuroprotecting effects through stimulating axonal regeneration and remyelination and decrement of lymphocyte infiltration. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A novel poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery: rheological, mucoadhesive and in vitro release properties.

PubMed

Mayol, Laura; Quaglia, Fabiana; Borzacchiello, Assunta; Ambrosio, Luigi; La Rotonda, Maria I

2008-09-01

The influence of hyaluronic acid (HA) on the gelation properties of poloxamers blends has been studied with the aim of engineering thermosensitive and mucoadhesive polymeric platforms for drug delivery. The gelation temperature (T(gel)), viscoelastic properties and mucoadhesive force of the systems were investigated and optimised by means of rheological analyses. Poloxamers micellar diameter was evaluated by photon correlation spectroscopy (PCS). Moreover in order to explore the feasibility of these platforms for drug delivery, the optimised systems were loaded with acyclovir and its release properties studied in vitro. By formulating poloxamers/HA platforms, at specific concentrations, it was possible to obtain a thermoreversible gel with a T(gel) close to body temperature. The addition of HA did not hamper the self assembling process of poloxamers just delaying the gelation temperature of few Celsius degrees. Furthermore, HA presence led to a strong increase of the poloxamer rheological properties thus indicating possible HA interactions with micelles through secondary bonds, such as hydrogen ones, which reinforce the gel structure. These interactions could also explain PCS results which show, in systems containing HA, aggregates with hydrodynamic diameters much higher than those of poloxamer micelles. Mucoadhesion experiments showed a rheological synergism between poloxamers/HA gels and mucin dispersion which led to a change of the flow behaviour from a quite Newtonian one of the separate solutions to a pseudoplastic one of their mixture. In vitro release experiments indicated that the optimised platform was able to prolong and control acyclovir release for more than 6h.

Dissolution and dissolution/permeation experiments for predicting systemic exposure following oral administration of the BCS class II drug clarithromycin.

PubMed

Kristin, Forner; René, Holm; Boontida, Morakul; Buraphacheep, Junyaprasert Varaporn; Maximilian, Ackermann; Johanna, Mazur; Peter, Langguth

2017-04-01

In order to save time and resources in early drug development, in vitro methods that correctly predict the formulation effect on oral drug absorption are necessary. The aim of this study was to 1) evaluate various BCS class II drug formulations with in vitro methods and in vivo in order to 2) determine which in vitro method best correlates with the in vivo results. Clarithromycin served as model compound in formulations with different particle sizes and content of excipients. The performed in vitro experiments were dissolution and dissolution/permeation experiments across two types of membrane, Caco-2 cells and excised rat intestinal sheets. The in vivo study was performed in rats. The oral absorption was enhanced by downsizing drug particles and by increasing the excipient concentration. This correlated strongly with the flux across Caco-2 cells but not with the other in vitro experiments. The insufficient correlation with the dissolution experiments can be partly explained by excipient caused problems during the filtration step. The very poor correlation of the in vivo data with the flux across excised rat intestinal sheets might be due to an artificially enlarged mucus layer ex vivo. In conclusion, downsizing BCS class II drug particles and the addition of surfactants enhanced the in vivo absorption, which was best depicted by dissolution/permeation experiments across Caco-2 cells. This setup is proposed as best model to predict the in vivo formulation effect. Also, this is the first study to evaluate the impact of the nature of the permeation membrane in dissolution/permeation experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of the addition of low rare earth elements (lanthanum, neodymium, cerium) on the biodegradation and biocompatibility of magnesium.

PubMed

Willbold, Elmar; Gu, Xuenan; Albert, Devon; Kalla, Katharina; Bobe, Katharina; Brauneis, Maria; Janning, Carla; Nellesen, Jens; Czayka, Wolfgang; Tillmann, Wolfgang; Zheng, Yufeng; Witte, Frank

2015-01-01

Rare earth elements are promising alloying element candidates for magnesium alloys used as biodegradable devices in biomedical applications. Rare earth elements have significant effects on the high temperature strength as well as the creep resistance of alloys and they improve magnesium corrosion resistance. We focused on lanthanum, neodymium and cerium to produce magnesium alloys with commonly used rare earth element concentrations. We showed that low concentrations of rare earth elements do not promote bone growth inside a 750 μm broad area around the implant. However, increased bone growth was observed at a greater distance from the degrading alloys. Clinically and histologically, the alloys and their corrosion products caused no systematic or local cytotoxicological effects. Using microtomography and in vitro experiments, we could show that the magnesium-rare earth element alloys showed low corrosion rates, both in in vitro and in vivo. The lanthanum- and cerium-containing alloys degraded at comparable rates, whereas the neodymium-containing alloy showed the lowest corrosion rates. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

PubMed

Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

2013-10-01

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Antioxidant Activity and Hepatoprotective Potential of Quercetin 7-Rhamnoside In Vitro and In Vivo.

PubMed

Huang, Zhi-Qiang; Chen, Pan; Su, Wei-Wei; Wang, Yong-Gang; Wu, Hao; Peng, Wei; Li, Pei-Bo

2018-05-16

Hypericum japonicum is traditionally used as a folk medicine to treat cholestasis and hepatitis. Quercetin 7-rhamnoside (Q7R) is one of the main flavonoid components of Hypericum japonicum and has been rarely studied. The aim of the present study was to evaluate the antioxidant activity and hepatoprotective potential of Q7R. In the in vitro experiments, DPPH, ABTS and ferric reducing antioxidant power (FRAP) assays were first performed to assess the antioxidant properties of Q7R, and then a H₂O₂-induced oxidative damage cellular model was used to determine the cytoprotective and antioxidant properties of Q7R in human liver L-02 cells. In the in vivo experiment, the hepatoprotective activity of Q7R was evaluated by carbon tetrachloride (CCl₄)-induced liver damage model in mice. The results of the three in vitro assays (DPPH, ABTS and FRAP) demonstrated that Q7R significantly exhibited antioxidant activity. The cell experiment results showed that Q7R possessed cytoprotective and antioxidant effects on H₂O₂-treated L-02 cells. In the in vivo experiments, Q7R suppressed the up-regulation of serum activities of ALT, AST, LDH and triglyceride (TG) levels with dose-dependency. Q7R down-regulated the production of MDA and increased the hepatic GSH content and antioxidant enzymes CAT activities. Hepatic morphological analysis was also performed to confirm the biochemical changes. In summary, these results suggested that Q7R could be considered as a potential source of natural antioxidants, and may become a promising candidate for the treatment of liver injury in the future.

Use of in-vitro experimental results to model in-situ experiments: bio-denitrification under geological disposal conditions.

PubMed

Masuda, Kaoru; Murakami, Hiroshi; Kurimoto, Yoshitaka; Kato, Osamu; Kato, Ko; Honda, Akira

2013-01-01

Some of the low level radioactive wastes from reprocessing of spent nuclear fuels contain nitrates. Nitrates can be present in the form of soluble salts and can be reduced by various reactions. Among them, reduction by metal compounds and microorganisms seems to be important in the underground repository. Reduction by microorganism is more important in near field area than inside the repository because high pH and extremely high salt concentration would prevent microorganism activities. In the near field, pH is more moderate (pH is around 8) and salt concentration is lower. However, the electron donor may be limited there and it might be the control factor for microorganism's denitrification activities. In this study, in-vitro experiments of the nitrate reduction reaction were conducted using model organic materials purported to exist in underground conditions relevant to geological disposal. Two kinds of organic materials were selected. A super plasticizer was selected as being representative of the geological disposal system and humic acid was selected as being representative of pre-existing organic materials in the bedrock. Nitrates were reduced almost to N2 gas in the existence of super plasticizer. In the case of humic acids, although nitrates were reduced, the rate was much lower and, in this case, dead organism was used as an electron donor instead of humic acids. A reaction model was developed based on the in-vitro experiments and verified by running simulations against data obtained from in-situ experiments using actual groundwaters and microorganisms. The simulation showed a good correlation with the experimental data and contributes to the understanding of microbially mediated denitrification in geological disposal systems.

Development of ligustrazine hydrochloride carboxymethyl chitosan and collagen microspheres: Formulation optimization, characterization, and vitro release

PubMed Central

Lin, Qiang; Huo, Qing; Qin, Yingzhe; Zhao, Zhuo; Tao, Fengyun

2017-01-01

ABSTRACT This study investigates the preparation of ligustrazine hydrochloride carboxymethyl chitosan and collagen microspheres. This experiment investigates effects of the ratio of carboxymethyl chitosan and collagen blend, water to oil ratio, stirring speed, and other factors on the microsphere properties. The experiment had the following conditions: a 1:2 proportion of carboxymethyl chitosan and collagen, a 1:2 proportion of drugs and materials, a 5:1 proportion of oil phase and water phase, 0.5% of span80, a 600r/min stirring speed, 3 ml of a cross-linking agent, 3 h of cross-linking curing, 1.25 ± 0.05 mm diameter LTH microcapsules, a 54.08% envelop rate, and a 14.16% carrier rate. The microspheres release rate reached 66% within 1 h, then steadily released within 5 h in vitro. The experimental results showed that the ligustrazine hydrochloride microsphere production process was stable and exhibited a good release effect compared with other ligustrazine hydrochloride tablets and pills. PMID:27689792

Harnessing the landscape of microbial culture media to predict new organism–media pairings

PubMed Central

Oberhardt, Matthew A.; Zarecki, Raphy; Gronow, Sabine; Lang, Elke; Klenk, Hans-Peter; Gophna, Uri; Ruppin, Eytan

2015-01-01

Culturing microorganisms is a critical step in understanding and utilizing microbial life. Here we map the landscape of existing culture media by extracting natural-language media recipes into a Known Media Database (KOMODO), which includes >18,000 strain–media combinations, >3300 media variants and compound concentrations (the entire collection of the Leibniz Institute DSMZ repository). Using KOMODO, we show that although media are usually tuned for individual strains using biologically common salts, trace metals and vitamins/cofactors are the most differentiating components between defined media of strains within a genus. We leverage KOMODO to predict new organism–media pairings using a transitivity property (74% growth in new in vitro experiments) and a phylogeny-based collaborative filtering tool (83% growth in new in vitro experiments and stronger growth on predicted well-scored versus poorly scored media). These resources are integrated into a web-based platform that predicts media given an organism's 16S rDNA sequence, facilitating future cultivation efforts. PMID:26460590

Non-animal approaches for toxicokinetics in risk evaluations of food chemicals.

PubMed

Punt, Ans; Peijnenburg, Ad A C M; Hoogenboom, Ron L A P; Bouwmeester, Hans

2017-01-01

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs versus that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics, thereby increasing regulatory acceptance of non-animal kinetic data.

The in vitro isolated whole guinea pig brain as a model to study epileptiform activity patterns.

PubMed

de Curtis, Marco; Librizzi, Laura; Uva, Laura

2016-02-15

Research on ictogenesis is based on the study of activity between seizures and during seizures in animal models of epilepsy (chronic condition) or in in vitro slices obtained from naïve non-epileptic brains after treatment with pro-convulsive drugs, manipulations of the extracellular medium and specific stimulation protocols. The in vitro isolated guinea pig brain retains the functional connectivity between brain structures and maintains interactions between neuronal, glial and vascular compartments. It is a close-to-in vivo preparation that offers experimental advantages not achieved with the use of other experimental models. Neurophysiological and imaging techniques can be utilized in this preparation to study brain activity during and between seizures induced by pharmacological or functional manipulations. Cellular and network determinants of interictal and ictal discharges that reproduce abnormal patterns observed in human focal epilepsies and the associated changes in extracellular ion and blood-brain permeability can be identified and analyzed in the isolated guinea pig brain. Ictal and interictal patterns recorded in in vitro slices may show substantial differences from seizure activity recorded in vivo due to slicing procedure itself. The isolated guinea pig brain maintained in vitro by arterial perfusion combines the typical facilitated access of in vitro preparations, that are difficult to approach during in vivo experiments, with the preservation of larger neuronal networks. The in vitro whole isolated guinea pig brain preparation offers an unique experimental model to study systemic and neurovascular changes during ictogenesis. Published by Elsevier B.V.

Effect of medium-chain triglycerides on the release behavior of Endostar encapsulated PLGA microspheres.

PubMed

Meng, Boyu; Li, Ling; Hua, Su; Wang, Qingsong; Liu, Chunhui; Xu, Xiangyang; Yin, Xiaojin

2010-09-15

The incomplete release of Endostar from PLGA microspheres was observed in our previous study. In the present study, we focused on the effect of medium-chain triglycerides (MCT) on the in vitro/in vivo release behavior of Endostar encapsulated PLGA microspheres, which were prepared by a water-in-oil-in-water (W/O/W) double-emulsion method with or without MCT. The in vitro accumulated release of Endostar from microspheres co-encapsulated with 30% MCT was found to be 79.04% after a 30-day incubation period in PBS (pH 7.4) at 37 degrees C. However, the accumulated release of Endostar from MCT-free microspheres was found to be only 32.22%. Pouches containing Endostar encapsulated PLGA microspheres were implanted subcutaneously in rats. The effect of MCT on the in vivo release showed a similar trend to the in vitro release. After 30 days, only 9.87% of the total encapsulated Endostar was retained in microspheres co-encapsulated with 30% MCT, while 42.25% of Endostar was retained in MCT-free microspheres. The co-encapsulation of MCT provided the microspheres with a porous surface, which significantly improved the in vitro/in vivo release of Endostar from PLGA microspheres. In addition, in vitro experiments showed that MCT co-encapsulated PLGA microspheres had more inter-connected pores, faster degradation of PLGA, and faster swelling of microspheres, which helped to explain the mechanism of the effect of MCT on improving the release of Endostar from PLGA microspheres. Copyright 2010 Elsevier B.V. All rights reserved.

Prevalidation of in vitro continuous flow exposure systems as alternatives to in vivo inhalation safety evaluation experimentations: outcome from MAAPHRI-PCRD5 research program.

PubMed

Morin, Jean-Paul; Hasson, Virginie; Fall, Mamadou; Papaioanou, Eleni; Preterre, David; Gouriou, Frantz; Keravec, Veronika; Konstandopoulos, Athanasios; Dionnet, Frédéric

2008-06-01

Diesel engine emission aerosol-induced toxicity patterns were compared using both in vitro (organotypic cultures of lung tissue) and in vivo experimentations mimicking the inhalation situation with continuous aerosol flow exposure designs. Using liquid media resuspended diesel particles, we show that toxic response pattern is influenced by the presence of tensioactive agent in the medium which alter particle-borne pollutant bioavailability. Using continuous aerosol exposure in vitro, we show that with high sulfur fuel (300ppm) in the absence of oxidation catalysis, particulate matter was the main toxic component triggering DNA damage and systemic inflammation, while a very limited oxidant stress was evidenced. In contrast, with ultra-low sulfur fuel in the presence of strong diesel oxidation catalysis, the specific role of particulate matter is no longer evidenced and the gas phase then becomes the major component triggering strong oxidant stress, increased NO(2) being the most probable trigger. In vivo, plasma tumor necrosis factor alpha (TNFalpha), lung superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activity levels varied in agreement with in vitro observations. Diesel emission treatment with oxycat provokes a marked systemic oxidant stress. Again NO(2) proved to account for a major part of these impacts. In conclusion, similar anti-oxidant responses were observed in in vitro and in vivo experiments after diesel emission aerosol continuous flow exposures. The lung slice organotypic culture model-exposed complex aerosol appears to be a very valuable alternative to in vivo inhalation toxicology experimentations in rodents.

In vitro blood and fibroblast responses to BisGMA-TEGDMA/bioactive glass composite implants.

PubMed

Abdulmajeed, Aous A; Kokkari, Anne K; Käpylä, Jarmo; Massera, Jonathan; Hupa, Leena; Vallittu, Pekka K; Närhi, Timo O

2014-01-01

This in vitro study was designed to evaluate both blood and human gingival fibroblast responses to bisphenol A-glycidyl methacrylate-triethyleneglycol dimethacrylate (BisGMA-TEGDMA)/bioactive glass (BAG) composite, aimed to be used as composite implant abutment surface modifier. Three different types of substrates were investigated: (a) plain polymer (BisGMA 50 wt%-TEGDMA 50 wt%), (b) BAG-composite (50 wt% polymer + 50 wt% fraction of BAG-particles, <50 μm), and (c) plain BAG plates (100 wt% BAG). The blood response, including the blood-clotting ability and platelet adhesion morphology were evaluated. Human gingival fibroblasts were plated and cultured on the experimental substrates for up to 10 days, then the cell proliferation rate was assessed using AlamarBlue assay™. The BAG-composite and plain BAG substrates had a shorter clotting time than plain polymer substrates. Platelet activation and aggregation were most extensive, qualitatively, on BAG-composite. Analysis of the normalized cell proliferation rate on the different surfaces showed some variations throughout the experiment, however, by day 10 the BAG-composite substrate showed the highest (P < 0.001) cell proliferation rate. In conclusion, the presence of exposed BAG-particles enhances fibroblast and blood responses on composite surfaces in vitro.

Ultrasound assisted extraction of polysaccharides from Lentinus edodes and its anti-hepatitis B activity in vitro.

PubMed

Zhao, Yong-Ming; Yang, Jian-Ming; Liu, Ying-Hui; Zhao, Ming; Wang, Jin

2018-02-01

The aim of this study was to optimize the extraction process of polysaccharides from the fruiting bodies of Lentinus edodes and investigate its anti-hepatitis B virus activity. The extracting parameters including ultrasonic power (240-320W), extraction temperature (40-60°C) and extraction time (15-25min) was optimized by using three-variable-three-level Box-Behnken design based on the single-factor experiments. Data analysis results showed that the optimal conditions for extracting LEPs were an extraction temperature of 45°C, extraction time of 21min and ultrasonic power of 290W. Under these optimal conditions, the experimental yield of LEPs was 9.75%, a 1.62-fold increase compared with conventional heat water extraction (HWE). In addition, crude polysaccharides were purified to obtain two fractions (LEP-1 and LEP-2). Chemical analysis showed that these components were rich in glucose, arabinose and mannose. Furthermore, HepG2.2.15 cells were used as in vitro models to evaluate their anti-hepatitis B virus (HBV) activity. The results suggest that LEPs possesses potent anti-HBV activity in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

MICRONUCLEI IN BINUCLEATED LYMPHOCYTES OF MICE FOLLOWING EXPOSURE TO GAMMA RADIATION

EPA Science Inventory

Experiments were designed to investigate the induction of micronuclei (MN) in mouse peripheral blood lymphocytes (PBLs) after in vitro or in vivo exposure to 60Co gamma radiation. or the in vitro experiments, 4 ml of blood from male C57BL/6J mice were either irradiated in 6 ml Fa...

"In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

ERIC Educational Resources Information Center

Higgins, Pamela J.

2005-01-01

This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

Integration of Vibrio vulnificus into Marine Aggregates and Its Subsequent Uptake by Crassostrea virginica Oysters

PubMed Central

Froelich, Brett; Ayrapetyan, Mesrop

2013-01-01

Marine aggregates are naturally forming conglomerations of larvacean houses, phytoplankton, microbes, and inorganics adhered together by exocellular polymers. In this study, we show in vitro that the bacterial pathogen Vibrio vulnificus can be concentrated into laboratory-generated aggregates from surrounding water. We further show that environmental (E-genotype) strains exhibit significantly more integration into these aggregates than clinical (C-genotype) strains. Experiments where marine aggregates with attached V. vulnificus cells were fed to oysters (Crassostrea virginica) resulted in greater uptake of both C and E types than nonaggregated controls. When C- and E-genotype strains were cocultured in competitive experiments, the aggregated E-genotype strains exhibited significantly greater uptake by oyster than the C-genotype strains. PMID:23263962

Electrochemistry-mass spectrometry for in-vitro determination of selected chemotherapeutics and their electrochemical products in comparison to in-vivo approach.

PubMed

Szultka-Mlynska, Malgorzata; Buszewski, Boguslaw

2016-11-01

Chemotherapeutics are among the most frequently prescribed medications in modern medicine. They are widely prescribed; however, problems with organisms developing resistance to these drugs means that their efficacy may be lost, so care should be taken to avoid unnecessary prescription. It is therefore of great interest to study the detailed metabolism of these biologically active compounds. This study aimed at developing an efficient analytical protocol for the determination of in-vitro electrochemical products of selected antibiotic drugs (amoxicillin, cefotaxime, fluconazole, linezolid, metronidazole and moxifloxacin). Combination of electrochemistry (EC) and mass spectrometry (MS) was applied for the in-vitro determination of the studied antibiotics and their electrochemical products. To identify the structure of the detected electrochemical products, MS/MS experiments were performed. This was one of the first applications of the EC system for generation of electrochemical products produced from antibiotic drugs. Adjustment of appropriate conditions and such parameters as the potential value, mobile phase (pH), working electrode and temperature had significant influence on electrochemical simulations and the creation of selected derivatives. Consequently, several working electrodes were evaluated for this purpose. In most of the studied cases, mainly two types of products were observed. One corresponded to an increase in mass by 14Da, which can be explained by a process consisting of oxidation (+16 m/z) and dehydrogenation (-2 m/z); The second in turn showed mass reduction by 14Da, which can be attributed to the loss of -CH2 as a result of N-demethylation. The performed experiments consisted of two stages: electrochemical oxidation of the analyzed samples (phase I of metabolic transformation), and addition of glutathione (GSH) for follow-up reactions (phase II conjunction). The electrochemical results were compared to in-vivo experiments by analyzing urine samples from patients after antibiotic drugs have been administered.. Overall, the comparison of electrochemistry to in-vivo experiments shows the high potential of EC-MS as a fast analytical tool in the prediction of electrochemical conversion that could be applied to therapeutic drug monitoring and pharmacokinetic studies as well. Copyright © 2016 Elsevier B.V. All rights reserved.

Antihyperglycemic effect of a natural chicoric acid extract of chicory (Cichorium intybus L.): a comparative in vitro study with the effects of caffeic and ferulic acids.

PubMed

Azay-Milhau, Jacqueline; Ferrare, Karine; Leroy, Jeremy; Aubaterre, Jordan; Tournier, Michel; Lajoix, Anne-Dominique; Tousch, Didier

2013-11-25

In Eurasia folk medicine, roots of chicory (Cichorium intybus L.) have been reported to exert antidiabetic benefits. In vitro, a natural chicoric acid extract (NCRAE) from Cichorium intybus root has been shown to increase insulin secretion by pancreatic β-cells and glucose uptake by muscle cells. In vitro experiments were designed to compare the effects of two hydroxycinnamic acids, caffeic and ferulic acids, to those obtained with NCRAE (50 and 100 µg.mL(-1)) on the three major tissues implicated in glycemic regulation (pancreas, muscle and liver). In vivo experiments were performed in Wistar rats submitted to a daily intraperitoneal injection of NCRAE (3, 15 or 30 mg kg(-1)) for 4 days. On the fourth day, an intraperitoneal glucose tolerance test (IPGTT; 1 g kg(-1)) was carried out. Our results show that the three compounds we used are able each to induce an original response. Caffeic acid mainly promotes a decrease in hepatic glycogenolysis. Ferulic acid elicits a clear increase of insulin release and a reduction of hepatic glycogenolysis. However, this compound induces an inhibition of muscle glucose uptake. NCRAE provokes an increase of insulin release and glucose uptake without any effect on hepatic glycogenolysis. We could also show that none of these compounds implicates hepatic glucose 6-phosphatase in contrast to chlorogenic acid, known as an inhibitor of glucose 6-phosphatase and which is able to decrease glucose output from hepatocytes. Our results point out that NCRAE is able to decrease blood glucose without any effect hepatic effect. Our in vivo experiments bring evidence that 4 daily IP administrations of NCRAE improve IP glucose tolerance in a dose-dependent manner and mainly via an insulin sensitizing effect. We conclude that NCRAE presents an antihyperglycemic effect essentially due to a peripheral effect on muscle glucose uptake. © 2013 Elsevier Ireland Ltd. All rights reserved.

Combined photothermal and photodynamic therapy delivered by PEGylated MoS2 nanosheets

NASA Astrophysics Data System (ADS)

Liu, Teng; Wang, Chao; Cui, Wei; Gong, Hua; Liang, Chao; Shi, Xiaoze; Li, Zhiwei; Sun, Baoquan; Liu, Zhuang

2014-09-01

Single- or few-layered transitional metal dichalcogenides, as a new genus of two-dimensional nanomaterials, have attracted tremendous attention in recent years, owing to their various intriguing properties. In this study, chemically exfoliated MoS2 nanosheets are modified with lipoic acid-terminated polyethylene glycol (LA-PEG), obtaining PEGylated MoS2 (MoS2-PEG) with high stability in physiological solutions and no obvious toxicity. Taking advantage of its ultra-high surface area, the obtained MoS2-PEG is able to load a photodynamic agent, chlorin e6 (Ce6), by physical adsorption. In vitro experiments reveal that Ce6 after being loaded on MoS2-PEG shows remarkably increased cellular uptake and thus significantly enhanced photodynamic therapeutic efficiency. Utilizing the strong, near-infrared (NIR) absorbance of the MoS2 nanosheets, we further demonstrate photothermally enhanced photodynamic therapy using Ce6-loaded MoS2-PEG for synergistic cancer killing, in both in vitro cellular and in vivo animal experiments. Our study presents a new type of multifunctional nanocarrier for the delivery of photodynamic therapy, which, if combined with photothermal therapy, appears to be an effective therapeutic approach for cancer treatment.Single- or few-layered transitional metal dichalcogenides, as a new genus of two-dimensional nanomaterials, have attracted tremendous attention in recent years, owing to their various intriguing properties. In this study, chemically exfoliated MoS2 nanosheets are modified with lipoic acid-terminated polyethylene glycol (LA-PEG), obtaining PEGylated MoS2 (MoS2-PEG) with high stability in physiological solutions and no obvious toxicity. Taking advantage of its ultra-high surface area, the obtained MoS2-PEG is able to load a photodynamic agent, chlorin e6 (Ce6), by physical adsorption. In vitro experiments reveal that Ce6 after being loaded on MoS2-PEG shows remarkably increased cellular uptake and thus significantly enhanced photodynamic therapeutic efficiency. Utilizing the strong, near-infrared (NIR) absorbance of the MoS2 nanosheets, we further demonstrate photothermally enhanced photodynamic therapy using Ce6-loaded MoS2-PEG for synergistic cancer killing, in both in vitro cellular and in vivo animal experiments. Our study presents a new type of multifunctional nanocarrier for the delivery of photodynamic therapy, which, if combined with photothermal therapy, appears to be an effective therapeutic approach for cancer treatment. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03753g

Supramolecular nanofibers of triamcinolone acetonide for uveitis therapy

NASA Astrophysics Data System (ADS)

Li, Xingyi; Wang, Yuqin; Yang, Chengbiao; Shi, Shuai; Jin, Ling; Luo, Zichao; Yu, Jing; Zhang, Zhaoliang; Yang, Zhimou; Chen, Hao

2014-11-01

Supramolecular nanofibers of prodrugs hold advantages for drug release due to their high drug payload, sustained and constant drug release behavior, and stimuli responsiveness. In this study, we report on a supramolecular hydrogel mainly formed by a clinically used drug triamcinolone acetonide (TA). Such a hydrogel could only be prepared via an ester bond hydrolysis process from its prodrug of succinated triamcinolone acetonide (STA). The resulting hydrogel could constantly release TA in the in vitro release experiment. The TA hydrogel possessed an excellent transscleral penetration ability, as evaluated by the in vitro transscleral transport study. The developed TA hydrogel also exhibited a great ocular compatibility in rats, as indicated by the optical coherence tomography (OCT) images, HE observation, and glial fibrillary acidic protein (GFAP) and vimentin immuno-staining assays of the retinas. Our TA hydrogel showed a decreased efficacy to inhibit ocular inflammation in the rat's experiment autoimmune uveitis (EAU) model compared to the commercial TA suspension (Transton®), but without causing complications such as high intraocular pressure and cataracts. These promising properties of the hydrogel indicated its great potential for the treatment of eye diseases.Supramolecular nanofibers of prodrugs hold advantages for drug release due to their high drug payload, sustained and constant drug release behavior, and stimuli responsiveness. In this study, we report on a supramolecular hydrogel mainly formed by a clinically used drug triamcinolone acetonide (TA). Such a hydrogel could only be prepared via an ester bond hydrolysis process from its prodrug of succinated triamcinolone acetonide (STA). The resulting hydrogel could constantly release TA in the in vitro release experiment. The TA hydrogel possessed an excellent transscleral penetration ability, as evaluated by the in vitro transscleral transport study. The developed TA hydrogel also exhibited a great ocular compatibility in rats, as indicated by the optical coherence tomography (OCT) images, HE observation, and glial fibrillary acidic protein (GFAP) and vimentin immuno-staining assays of the retinas. Our TA hydrogel showed a decreased efficacy to inhibit ocular inflammation in the rat's experiment autoimmune uveitis (EAU) model compared to the commercial TA suspension (Transton®), but without causing complications such as high intraocular pressure and cataracts. These promising properties of the hydrogel indicated its great potential for the treatment of eye diseases. Electronic supplementary information (ESI) available: Synthesis and characterization of the STA agent. SEM image of TA suspension (Transton®). See DOI: 10.1039/c4nr04761c

In vivo relevance of two critical levels for NAD(P)H:quinone oxidoreductase (NQO1)-mediated cellular protection against electrophile toxicity found in vitro.

PubMed

de Haan, Laura H J; Pot, Gerda K; Aarts, Jac M M J G; Rietjens, Ivonne M C M; Alink, Gerrit M

2006-08-01

NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones is suggested to be involved in cancer prevention. In the present study, using transfected CHO cells, it was demonstrated that the relation between NQO1 activity and the resulting protection against the cytotoxicity of menadione shows a steep dose-response curve revealing a 'lower protection threshold' of 0.5mumol DCPIP/min/mg protein and an 'upper protection threshold' at 1mumol DCPIP/min/mg protein. In an additional in vivo experiment it was investigated how both in vitro critical activity levels of NQO1, relate to NQO1 activities in mice and man, either without or upon induction of the enzyme by butylated hydroxyanisol (BHA) or indole-3-carbinol (I(3)C). Data from an experiment with CD1 mice revealed that base-line NQO1 levels in liver, kidney, small intestine, colon and lung are generally below the observed 'lower protection threshold' in vitro, this also holds for most human tissue S-9 samples. To achieve NQO1 levels above this 'lower protection threshold' will require 5-20 fold NQO1 induction. Discussion focuses on the relevance of the in vitro NQO1 activity thresholds for the in vivo situation. We conclude that increased protection against menadione toxicity can probably not be achieved by NQO1 induction but should be achieved by other mechanisms. Whether this conclusion also holds for other electrophiles and the in vivo situation awaits further definition of their NQO1 protection thresholds.

Continuous "in vitro" Evolution of a Ribozyme Ligase: A Model Experiment for the Evolution of a Biomolecule

ERIC Educational Resources Information Center

Ledbetter, Michael P.; Hwang, Tony W.; Stovall, Gwendolyn M.; Ellington, Andrew D.

2013-01-01

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. "In vitro" directed evolution (IVDE) frequently serves as a model system for the study of Darwinian…

MEDIA SERUM LEVELS AND IN VITRO HEPATIC ABSORPTION OF LINDANE

EPA Science Inventory

High plasma protein binding is known to reduce the tissue uptake of chemicals in vivo, but the extent of its importance in vitro is less clear. Experiments were conducted to determine the cellular uptake of lindane in vitro under different conditions. Lindane was selected because...

Active Curved Polymers Form Vortex Patterns on Membranes.

PubMed

Denk, Jonas; Huber, Lorenz; Reithmann, Emanuel; Frey, Erwin

2016-04-29

Recent in vitro experiments with FtsZ polymers show self-organization into different dynamic patterns, including structures reminiscent of the bacterial Z ring. We model FtsZ polymers as active particles moving along chiral, circular paths by Brownian dynamics simulations and a Boltzmann approach. Our two conceptually different methods point to a generic phase behavior. At intermediate particle densities, we find self-organization into vortex structures including closed rings. Moreover, we show that the dynamics at the onset of pattern formation is described by a generalized complex Ginzburg-Landau equation.

An acid-free water-born quaternized chitosan/montmorillonite loaded into an innovative ultra-fine bead-free water-born nanocomposite nanofibrous scaffold; in vitro and in vivo approaches.

PubMed

Dastjerdi, Roya; Sharafi, Mahsa; Kabiri, Kourosh; Mivehi, Leila; Samadikuchaksaraei, Ali

2017-07-26

An acid-free water-born chitosan derivative/montmorillonite has been successfully synthesized. A natural-based biopolymer, N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride, was synthesized, and its structure confirmed by Fourier transform infrared microscopy and conductometric titration. It was applied to the cationic ion-exchange reaction of montmorillonite. Then, the synthesized materials were used to produce water-born composite scaffolds for tissue engineering applications and formed an ultra-fine bead-free multicomponent nanofibrous scaffold. The scaffold was subjected to in vitro and in vivo investigations. The effects of both acidic and neutral reaction media on the efficiency of the cationic ion-exchange reaction of montmorillonite were investigated. A mechanism has been suggested for the more efficient cationic ion-exchange reaction achieved in the absence of the acid. In in vitro studies, the modified montmorillonite showed synergistic biocompatibility and cell growth with enhanced bioactivity compared to unmodified clay and even chitosan and the chitosan derivative. Scanning electron microscopy showed ultra-fine bead-free nanocomposite nanofibers. Improved biocompatibility, cell attachment, and cell growth were observed for the nanofibrous scaffolds compared to the individual components. In vivo experiments showed complete restoration of a critical-sized full-thickness wound without infection in 21 d. The technique provides a guideline to achieve chitosan nanofibrous morphology for multifunctional biomedical applications.

In Vitro-In Vivo Extrapolation of Metabolism- and Transporter-Mediated Drug-Drug Interactions-Overview of Basic Prediction Methods.

PubMed

Yoshida, Kenta; Zhao, Ping; Zhang, Lei; Abernethy, Darrell R; Rekić, Dinko; Reynolds, Kellie S; Galetin, Aleksandra; Huang, Shiew-Mei

2017-09-01

Evaluation of drug-drug interaction (DDI) risk is vital to establish benefit-risk profiles of investigational new drugs during drug development. In vitro experiments are routinely conducted as an important first step to assess metabolism- and transporter-mediated DDI potential of investigational new drugs. Results from these experiments are interpreted, often with the aid of in vitro-in vivo extrapolation methods, to determine whether and how DDI should be evaluated clinically to provide the basis for proper DDI management strategies, including dosing recommendations, alternative therapies, or contraindications under various DDI scenarios and in different patient population. This article provides an overview of currently available in vitro experimental systems and basic in vitro-in vivo extrapolation methodologies for metabolism- and transporter-mediated DDIs. Published by Elsevier Inc.

Marine Collagen Peptides from the Skin of Nile Tilapia (Oreochromis niloticus): Characterization and Wound Healing Evaluation

PubMed Central

Hu, Zhang; Yang, Ping; Zhou, Chunxia; Li, Sidong; Hong, Pengzhi

2017-01-01

Burns can cause tremendous economic problems associated with irreparable harm to patients and their families. To characterize marine collagen peptides (MCPs) from the skin of Nile tilapia (Oreochromis niloticus), molecular weight distribution and amino acid composition of MCPs were determined, and Fourier transform infrared spectroscopy (FTIR) was used to analyze the chemical structure. Meanwhile, to evaluate the wound healing activity, in vitro and in vivo experiments were carried out. The results showed that MCPs prepared from the skin of Nile tilapia by composite enzymatic hydrolysis were composed of polypeptides with different molecular weights and the contents of polypeptides with molecular weights of less than 5 kDa accounted for 99.14%. From the amino acid composition, the majority of residues, accounting for over 58% of the total residues in MCPs, were hydrophilic. FTIR indicated that the main molecular conformations inside MCPs were random coil. In vitro scratch assay showed that there were significant effects on the scratch closure by the treatment of MCPs with the concentration of 50.0 μg/mL. In the experiments of deep partial-thickness scald wound in rabbits, MCPs could enhance the process of wound healing. Therefore, MCPs from the skin of Nile tilapia (O. niloticus) have promising applications in wound care. PMID:28358307

Marine Collagen Peptides from the Skin of Nile Tilapia (Oreochromis niloticus): Characterization and Wound Healing Evaluation.

PubMed

Hu, Zhang; Yang, Ping; Zhou, Chunxia; Li, Sidong; Hong, Pengzhi

2017-03-30

Burns can cause tremendous economic problems associated with irreparable harm to patients and their families. To characterize marine collagen peptides (MCPs) from the skin of Nile tilapia ( Oreochromis niloticus ), molecular weight distribution and amino acid composition of MCPs were determined, and Fourier transform infrared spectroscopy (FTIR) was used to analyze the chemical structure. Meanwhile, to evaluate the wound healing activity, in vitro and in vivo experiments were carried out. The results showed that MCPs prepared from the skin of Nile tilapia by composite enzymatic hydrolysis were composed of polypeptides with different molecular weights and the contents of polypeptides with molecular weights of less than 5 kDa accounted for 99.14%. From the amino acid composition, the majority of residues, accounting for over 58% of the total residues in MCPs, were hydrophilic. FTIR indicated that the main molecular conformations inside MCPs were random coil. In vitro scratch assay showed that there were significant effects on the scratch closure by the treatment of MCPs with the concentration of 50.0 μg/mL. In the experiments of deep partial-thickness scald wound in rabbits, MCPs could enhance the process of wound healing. Therefore, MCPs from the skin of Nile tilapia ( O. niloticus ) have promising applications in wound care.

[Preparation of curcumin-EC sustained-release composite particles by supercritical CO2 anti-solvent technology].

PubMed

Bai, Wei-li; Yan, Ting-yuan; Wang, Zhi-xiang; Huang, De-chun; Yan, Ting-xuan; Li, Ping

2015-01-01

Curcumin-ethyl-cellulose (EC) sustained-release composite particles were prepared by using supercritical CO2 anti-solvent technology. With drug loading and yield of inclusion complex as evaluation indexes, on the basis of single factor tests, orthogonal experimental design was used to optimize the preparation process of curcumin-EC sustained-release composite particles. The experiments such as drug loading, yield, particle size distribution, electron microscope analysis (SEM) , infrared spectrum (IR), differential scanning calorimetry (DSC) and in vitro dissolution were used to analyze the optimal process combination. The orthogonal experimental optimization process conditions were set as follows: crystallization temperature 45 degrees C, crystallization pressure 10 MPa, curcumin concentration 8 g x L(-1), solvent flow rate 0.9 mL x min(-1), and CO2 velocity 4 L x min(-1). Under the optimal conditions, the average drug loading and yield of curcumin-EC sustained-release composite particles were 33.01% and 83.97%, and the average particle size of the particles was 20.632 μm. IR and DSC analysis showed that curcumin might complex with EC. The experiments of in vitro dissolution showed that curcumin-EC composite particles had good sustained-release effect. Curcumin-EC sustained-release composite particles can be prepared by supercritical CO2 anti-solvent technology.

Design of experiments to optimize an in vitro cast to predict human nasal drug deposition.

PubMed

Shah, Samir A; Dickens, Colin J; Ward, David J; Banaszek, Anna A; George, Chris; Horodnik, Walter

2014-02-01

Previous studies showed nasal spray in vitro tests cannot predict in vivo deposition, pharmacokinetics, or pharmacodynamics. This challenge makes it difficult to assess deposition achieved with new technologies delivering to the therapeutically beneficial posterior nasal cavity. In this study, we determined best parameters for using a regionally divided nasal cast to predict deposition. Our study used a model suspension and a design of experiments to produce repeatable deposition results that mimic nasal deposition patterns of nasal suspensions from the literature. The seven-section (the nozzle locator, nasal vestibule, front turbinate, rear turbinate, olfactory region, nasopharynx, and throat filter) nylon nasal cast was based on computed tomography images of healthy humans. It was coated with a glycerol/Brij-35 solution to mimic mucus. After assembling and orienting, airflow was applied and nasal spray containing a model suspension was sprayed. After disassembling the cast, drug depositing in each section was assayed by HPLC. The success criteria for optimal settings were based on nine in vivo studies in the literature. The design of experiments included exploratory and half factorial screening experiments to identify variables affecting deposition (angles, airflow, and airflow time), optimization experiments, and then repeatability and reproducibility experiments. We found tilt angle and airflow time after actuation affected deposition the most. The optimized settings were flow rate of 16 L/min, postactuation flow time of 12 sec, a tilt angle of 23°, nozzle angles of 0°, and actuation speed of 5 cm/sec. Neither cast nor operator caused significant variation of results. We determined cast parameters to produce results resembling suspension nasal sprays in the literature. The results were repeatable and unaffected by operator or cast. These nasal spray parameters could be used to assess deposition from new devices or formulations. For human deposition studies using radiolabeled formulations, this cast could show that radiolabel deposition represents drug deposition. Our methods could also be used to optimize settings for other casts.

Micropropagation and Biomass Production of True-to-Type Stevia rebaudiana Bertoni.

PubMed

Modi, Arpan R; Sharma, Vikas; Patil, Ghanshyam; Singh, Amritpal S; Subhash, N; Kumar, Nitish

2016-01-01

Here we describe an efficient micropropagation protocol for Stevia rebaudiana Bertoni. We present experiments carried out to optimize the suitable media for in vitro shoot multiplication and root induction and to study the effect of culture vessel on shoot multiplication. Among all different media tested for in vitro shoot multiplication, hormone-free liquid medium is most suitable. The highest number of nodes per shoot (5.4) and length of shoot (4.76 cm) at 4 weeks after subculturing are observed when single node explants are placed on modified MS medium supplemented with 1 % sucrose and 0.7 % agar. The highest response of multiplication rate (9.56) is observed on half strength of macroelement of MS with full strength of microelement of MS and 170 mg/l KH2PO4, and 185 mg/l MgSO4 in plastic growth container. Further, RAPD marker analysis of in vitro-raised plants maintained their clonal fidelity and true-to-type without showing any somaclonal variation.

Dexpanthenol modulates gene expression in skin wound healing in vivo.

PubMed

Heise, R; Skazik, C; Marquardt, Y; Czaja, K; Sebastian, K; Kurschat, P; Gan, L; Denecke, B; Ekanayake-Bohlig, S; Wilhelm, K-P; Merk, H F; Baron, J M

2012-01-01

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1β, CYP1B1, CXCL1, CCL18 and KAP 4-2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts. Copyright © 2012 S. Karger AG, Basel.

In Vitro Virucidal and Virustatic Properties of the Crude Extract of Cynodon dactylon against Porcine Reproductive and Respiratory Syndrome Virus

PubMed Central

Khonghiran, Oapkun; Kunanoppadol, Suchaya; Potha, Teerapong; Chuammitri, Phongsakorn

2014-01-01

The in vitro virustatic and virucidal tests of the crude extract of Cynodon dactylon against infection with porcine reproductive and respiratory syndrome virus (PRRSV), a cause of major devastating pig disease, were described. Crude extract of C. dactylon was prepared for cytotoxicity on tissue-culture cells that were used to measure virustatic and virucidal activities against PRRSV. Crude extract of C. dactylon at 0.78 mg/mL showed no cytotoxicity on the cell line, and at that concentration significantly inhibited replication of PRRSV as early as 24 hours post infection (hpi). C. dactylon also inactivated PRRSV as determined by immunoperoxidase monolayer assay (IPMA) compared to the control experiments. In summary, the present study may be among the earliest studies to describe virustatic and virucidal activities of C. dactylon crude extract against PRRSV in vitro. Extracts of C. dactylon may be useful for PRRSV control and prevention on pig farms. PMID:24744959

Scientific Evidence of Rice By-Products for Cancer Prevention: Chemopreventive Properties of Waste Products from Rice Milling on Carcinogenesis In Vitro and In Vivo

PubMed Central

Tan, Bee Ling

2017-01-01

Cancer is a significant global health concern affecting men and women worldwide. Although current chemopreventive drugs could inhibit the growth of cancer cells, they exert many adverse side effects. Dietary factor plays a crucial role in the management of cancers and has drawn the attention of researchers to be used as an option to combat this disease. Both in vitro and in vivo studies showed that rice and its by-products display encouraging results in the prevention of this disease. The mechanism of anticancer effect is suggested partly through potentiation of bioactive compounds like vitamin E, phytic acid, γ-aminobutyric acid (GABA), γ-oryzanol, and phenolics. Nevertheless, the bioactivity of rice and its by-products is still incompletely understood. In this review, we present the findings from a preclinical study both in in vitro and in animal experiments on the promising role of rice by-products with focus on cancer prevention. PMID:28210630

Design, synthesis, and in vitro transfection biology of novel tocopherol based monocationic lipids: a structure-activity investigation.

PubMed

Kedika, Bhavani; Patri, Srilakshmi V

2011-01-27

Herein, we report on the design, synthesis, and in vitro gene delivery efficacies of five novel tocopherol based cationic lipids (1-5) in transfecting CHO, B16F10, A-549, and HepG2 cells. The in vitro gene transfer efficiencies of lipids (1-5) were evaluated by both β-galactosidase reporter gene expression and inverted fluorescent microscopic experiments. The results of the present structure-activity investigation convincingly demonstrate that the tocopherol based lipid with three hydroxyl groups in its headgroup region showed 4-fold better transfection efficiency than the commercial formulation. The results also demonstrate that these tocopherol based lipids may be targeted to liver. Transfection efficiency of all the relevant lipids was maintained even when the serum was present during the transfection conditions. The results indicated that the designed systems are quite capable of transferring the DNA into all four types of cells studied with low or no toxicity.

Apigenin inhibits African swine fever virus infection in vitro.

PubMed

Hakobyan, Astghik; Arabyan, Erik; Avetisyan, Aida; Abroyan, Liana; Hakobyan, Lina; Zakaryan, Hovakim

2016-12-01

African swine fever virus (ASFV) is one of the most devastating diseases of domestic pigs for which no effective vaccines are available. Flavonoids, natural products isolated from plants, have been reported to have significant in vitro and in vivo antiviral activity against different viruses. Here, we tested the antiviral effect of five flavonoids on the replication of ASFV in Vero cells. Our results showed a potent, dose-dependent anti-ASFV effect of apigenin in vitro. Time-of-addition experiments revealed that apigenin was highly effective at the early stages of infection. Apigenin reduced the ASFV yield by more than 99.99 % when it was added at 1 hpi. The antiviral activity of apigenin was further investigated by evaluation of ASFV protein synthesis and viral factories. This flavonoid inhibited ASFV-specific protein synthesis and viral factory formation. ASFV-infected cells continuously treated with apigenin did not display a cytopathic effect. Further studies addressing the use of apigenin in vivo are needed.

Scientific Evidence of Rice By-Products for Cancer Prevention: Chemopreventive Properties of Waste Products from Rice Milling on Carcinogenesis In Vitro and In Vivo.

PubMed

Tan, Bee Ling; Norhaizan, Mohd Esa

2017-01-01

Cancer is a significant global health concern affecting men and women worldwide. Although current chemopreventive drugs could inhibit the growth of cancer cells, they exert many adverse side effects. Dietary factor plays a crucial role in the management of cancers and has drawn the attention of researchers to be used as an option to combat this disease. Both in vitro and in vivo studies showed that rice and its by-products display encouraging results in the prevention of this disease. The mechanism of anticancer effect is suggested partly through potentiation of bioactive compounds like vitamin E, phytic acid, γ -aminobutyric acid (GABA), γ -oryzanol, and phenolics. Nevertheless, the bioactivity of rice and its by-products is still incompletely understood. In this review, we present the findings from a preclinical study both in in vitro and in animal experiments on the promising role of rice by-products with focus on cancer prevention.

Zinc attenuates forskolin-stimulated electrolyte secretion without involvement of the enteric nervous system in small intestinal epithelium from weaned piglets.

PubMed

Feng, Zike; Carlson, Dorthe; Poulsen, Hanne Damgaard

2006-11-01

In a previous study, we found that secretagogue-stimulated electrolyte secretion was attenuated by dietary and serosal zinc in piglet small intestinal epithelium in Ussing chambers. Several studies show that the enteric nervous system (ENS) is involved in regulation of electrolyte and/or fluid transport in intestinal epithelium from many species. The aim of the present study is to examine the mechanisms behind the attenuating effect of zinc on electrolyte secretion and to study whether the ENS is involved in this effect of zinc in vitro. Twenty-four piglets (six litters of four piglets) were allocated randomly to one of two dietary treatments consisting of a basic diet supplemented with 100 mg zinc/kg (Zn(100)) or 2500 mg zinc/kg (Zn(2500)), as ZnO. All the piglets were killed at 5-6 days after weaning and in vitro experiments with small intestinal epithelium in Ussing chambers were carried out. Furthermore, zinc, copper, alkaline phosphatase (AP) and metallothionein (MT) in mucosa, liver, and plasma were measured. These measurements showed that zinc status was increased in the Zn(2500) compared to the Zn(100) fed piglets. The in vitro studies did not confirm previous findings of attenuating effects of dietary zinc and zinc in vitro on the 5-HT induced secretion. But it showed that the addition of zinc at the serosal side attenuated the forskolin (FSK) (cAMP-dependent) induced ion secretion in epithelium from piglets fed with Zn(100) diet. Blocking the ENS with lidocaine or hexamethonium apparently slightly reduced this effect of zinc in vitro, but did not remove the effect of zinc. Consequently, it is suggested that zinc attenuates the cAMP dependent ion secretion mainly due to an effect on epithelial cells rather than affecting the mucosal neuronal pathway.

T cell regulation in microgravity - The current knowledge from in vitro experiments conducted in space, parabolic flights and ground-based facilities

NASA Astrophysics Data System (ADS)

Hauschild, Swantje; Tauber, Svantje; Lauber, Beatrice; Thiel, Cora S.; Layer, Liliana E.; Ullrich, Oliver

2014-11-01

Dating back to the Apollo and Skylab missions, it has been reported that astronauts suffered from bacterial and viral infections during space flight or after returning to Earth. Blood analyses revealed strongly reduced capability of human lymphocytes to become active upon mitogenic stimulation. Since then, a large number of in vitro studies on human immune cells have been conducted in space, in parabolic flights, and in ground-based facilities. It became obvious that microgravity affects cell morphology and important cellular functions. Observed changes include cell proliferation, the cytoskeleton, signal transduction and gene expression. This review gives an overview of the current knowledge of T cell regulation under altered gravity conditions obtained by in vitro studies with special emphasis on the cell culture conditions used. We propose that future in vitro experiments should follow rigorous standardized cell culture conditions, which allows better comparison of the results obtained in different flight- and ground-based experiment platforms.

Synthetic thrombus model for in vitro studies of laser thrombolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hermes, R.E.; Trajkovska, K.

1998-07-01

Laser thrombolysis is the controlled ablation of a thrombus (blood clot) blockage in a living arterial system. Theoretical modeling of the interaction of laser light with thrombi relies on the ability to perform in vitro experiments with well characterized surrogate materials. A synthetic thrombus formulation may offer more accurate results when compared to in vivo clinical experiments. The authors describe the development of new surrogate materials based on formulations incorporating chick egg, guar gum, modified food starch, and a laser light absorbing dye. The sound speed and physical consistency of the materials were very close to porcine (arterial) and humanmore » (venous) thrombi. Photographic and videotape recordings of pulsed dye laser ablation experiments under various experimental conditions were used to evaluate the new material as compared to in vitro tests with human (venous) thrombus. The characteristics of ablation and mass removal were similar to that of real thrombi, and therefore provide a more realistic model for in vitro laser thrombolysis when compared to gelatin.« less

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taxvig, Camilla, E-mail: camta@food.dtu.dk; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenousmore » metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.« less

Inhibitory effects of β,β-dimethylacrylshikonin on hepatocellular carcinoma in vitro and in vivo.

PubMed

Wu, Yi-ying; Wan, Li-hong; Zheng, Xiao-wei; Shao, Zhen-jun; Chen, Jian; Chen, Xia-jing; Liu, Li-tao; Kuang, Wen-juan; Tan, Xian-shu; Zhou, Li-ming

2012-05-01

β,β-Dimethylacrylshikonin is one of the most abundant naphthoquinones in the root extracts of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), which have been reported to have antitumor effects. This study evaluated the antiproliferative activity of β,β-dimethylacrylshikonin on human hepatocellular carcinoma (HCC) cells both in vitro and in vivo. In vitro, the MTT assay showed that β,β-dimethylacrylshikonin inhibited the proliferation of SMMC-7721 cells in both dose- and time-dependent manners with its 50% inhibitory concentration (IC(50) ) at 48 h being 15.01 ± 0.76 µg/mL. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and Hoechst staining detected the characteristics of cell apoptosis in β,β-dimethylacrylshikonin-treated cells and the apoptotic rates of treated groups were increased in a dose-dependent manner. Flow cytometric analysis revealed that β,β-dimethylacrylshikonin could block the cell cycle arrest at G2 phase. Furthermore, β,β-dimethylacrylshikonin down-regulated the mRNA and protein expression of Bcl-2 but up-regulated that of Bax. The cleaved caspase-3 protein was also detected in treated cells. The experiment in vivo showed that β,β-dimethylacrylshikonin significantly suppressed the growth of H(22) transplantable hepatoma, and induced the activation of caspase-3 determined by immunohistochemistry. The results indicate that β,β-dimethylacrylshikonin has significant antitumor effects on hepatocellular carcinoma both in vitro and in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

Photocontrolled reversible self-assembly of dodecamer nitrilase.

PubMed

Yu, Qiao; Wang, Yong; Zhao, Shengyun; Ren, Yuhong

2017-01-01

Naturally photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer. In this study, a method for the precise and reversible inducible self-assembly of dodecamer nitrilase in vivo (in Escherichia coli ) and in vitro (in a cell-free solution) was developed by means of the photoswitch-improved light-inducible dimer (iLID) system which could induce protein-protein dimerization. Nitrilase was fused with the photoswitch protein AsLOV2-SsrA to achieve the photocontrolled self-assembly of dodecamer nitrilase. The fusion protein self-assembled into a supramolecular assembly when illuminated at 470 nm. Scanning electron microscopy showed that the assembly formed a circular sheet structure. Self-assembly was also induced by light in E. coli . Dynamic light scattering and turbidity assay experiments showed that the assemblies formed within a few seconds under 470-nm light and completely disassembled within 5 min in the dark. Assembly and disassembly could be maintained for at least five cycles. Both in vitro and in vivo, the assemblies retained 90% of the initial activity of nitrilase and could be reused at least four times in vitro with 90% activity. An efficient method was developed for the photocontrolled assembly and disassembly of dodecamer nitrilase and for scaffold-free reversible self-assembly of multiple oligomeric enzymes in vivo and in vitro, providing new ideas and methods for immobilization of enzyme without carrier.

The anti-Malassezia furfur activity in vitro and in experimental dermatitis of six imidazole antifungal agents: bifonazole, clotrimazole, flutrimazole, ketoconazole, miconazole and sertaconazole.

PubMed

Van Gerven, F; Odds, F C

1995-01-01

Bifonazole, clotrimazole, flutrimazole, ketoconazole, miconazole and sertaconazole were tested for their activity against 23 isolates of Malassezia furfur by agar dilution in vitro. Topical formulations of the same agents were evaluated for efficacy against M. furfur skin infections in guinea pigs in vivo. The most potent inhibitor in vitro was ketoconazole (geometric mean minimum inhibitory concentration 0.51 microgram ml-1), followed by bifonazole (8.1 micrograms ml-1), then miconazole (14 micrograms ml-1), clotrimazole (15 micrograms ml-1) and flutrimazole (16 micrograms ml-1), with sertaconazole the least active (52 micrograms ml-1). In animal experiments involving three consecutive days of topical treatments, bifonazole 1% cream, clotrimazole 1% cream, flutrimazole 1% and 2% creams, ketoconazole 2% cream and shampoo and miconazole 2% cream all reduced M. furfur dermatitis lesion severity below that of untreated control animals; however, sertaconazole 2% gel and cream showed no reduction in lesion severity below control. The results confirm that ketoconazole is a more potent inhibitor of M. furfur in vitro than other topical antifungal agents of its class and suggest that sertaconazole is the least effective of such agents among those tested.

Selection of Lactic Acid Bacteria with Probiotic Potential Isolated from the Fermentation Process of "Cupuaçu" (Theobroma grandiflorum).

PubMed

Ornellas, Roberta Maria Santos; Santos, Tiza Teles; Arcucio, Leonardo Borges; Sandes, Sávio Henrique Cicco; Oliveira, Mayara Messias; Dias, Cristiano Villela; de Carvalho Silva, Samuel; Uetanabaro, Ana Paula Trovatti; Vinderola, Gabriel; Nicoli, Jacques Robert

2017-01-01

In the present study, nine lactic acid bacteria isolated from the fermentation process of "cupuaçu" (Theobroma grandiflorum) were selected for probiotic use. In vitro (resistance to gastrointestinal environment, in vitro antagonism and co-aggregation with pathogens) and in vivo (intestinal colonization and ex vivo antagonism in germ-free mice, cumulative mortality, translocation to liver and spleen, histopathological examination of liver and ileum and mRNA cytokine gene expression during an experimental infection with S. Typhimurium) assays were used. Among the nine Lactobacillus strains isolated from the "cupuaçu" fermentation, L. plantarum 81 and L. plantarum 90 were selected as potential probiotics based on better results obtained in in vitro evaluations (production of diffusible inhibitory compounds and co-aggregation) as well as in vivo experiments (resistance to gastrointestinal environment, ex vivo antagonism, higher survival after enteropathogen challenge, lower hepatic translocation of enteropathogen, lower histopathological lesions in ileum and liver and anti-inflammatory pattern of immunological response). Concluding, L. plantarum 81 and L. plantarum 90 showed in vitro and in vivo capacities for probiotic use through different mechanisms of protection and its origin would allow an easier adaptation in an alimentary matrix for its administration.

Pharmacokinetics and correlation between in vitro release and in vivo absorption of bio-adhesive pellets of panax notoginseng saponins.

PubMed

Li, Ying; Zhang, Yun; Zhu, Chun-Yan

2017-02-01

The present study was designed to prepare and compare bio-adhesive pellets of panax notoginseng saponins (PNS) with hydroxy propyl methyl cellulose (HPMC), chitosan, and chitosan : carbomer, explore the influence of different bio-adhesive materials on pharmacokinetics behaviors of PNSbio-adhesive pellets, and evaluate the correlation between in vivo absorption and in vitro release (IVIVC). In order to predict the in vivo concentration-time profile by the in vitro release data of bio-adhesive pellets, the release experiment was performed using the rotating basket method in pH 6.8 phosphate buffer. The PNS concentrations in rat plasma were analyzed by HPLC-MS-MS method and the relative bioavailability and other pharmacokinetic parameters were estimated using Kinetica4.4 pharmacokinetic software. Numerical deconvolution method was used to evaluate IVIVC. Our results indicated that, compared with ordinary pellets, PNS bio-adhesive pellets showed increased oral bioavailability by 1.45 to 3.20 times, increased C max , and extended MRT. What's more, the release behavior of drug in HPMC pellets was shown to follow a Fickian diffusion mechanism, a synergetic function of diffusion and skeleton corrosion. The in vitro release and the in vivo biological activity had a good correlation, demonstrating that the PNS bio-adhesive pellets had a better sustained release. Numerical deconvolution technique showed the advantage in evaluation of IVIVC for self-designed bio-adhesive pellets with HPMC. In conclusion, the in vitro release data of bio-adhesive pellets with HPMC can predict its concentration-time profile in vivo. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

In vitro fabrication of a tissue engineered human cardiovascular patch for future use in cardiovascular surgery.

PubMed

Yang, Chao; Sodian, Ralf; Fu, Ping; Lüders, Cora; Lemke, Thees; Du, Jing; Hübler, Michael; Weng, Yuguo; Meyer, Rudolf; Hetzer, Roland

2006-01-01

One approach to tissue engineering has been the development of in vitro conditions for the fabrication of functional cardiovascular structures intended for implantation. In this experiment, we developed a pulsatile flow system that provides biochemical and biomechanical signals in order to regulate autologous, human patch-tissue development in vitro. We constructed a biodegradable patch scaffold from porous poly-4-hydroxy-butyrate (P4HB; pore size 80 to 150 microm). The scaffold was seeded with pediatric aortic cells. The cell-seeded patch constructs were placed in a self-developed bioreactor for 7 days to observe potential tissue formation under dynamic cell culture conditions. As a control, cell-seeded scaffolds were not conditioned in the bioreactor system. After maturation in vitro, the analysis of the tissue engineered constructs included biochemical, biomechanical, morphologic, and immunohistochemical examination. Macroscopically, all tissue engineered constructs were covered by cells. After conditioning in the bioreactor, the cells were mostly viable, had grown into the pores, and had formed tissue on the patch construct. Electron microscopy showed confluent smooth surfaces. Additionally, we demonstrated the capacity to generate collagen and elastin under in vitro pulsatile flow conditions in biochemical examination. Biomechanical testing showed mechanical properties of the tissue engineered human patch tissue without any statistical differences in strength or resistance to stretch between the static controls and the conditioned patches. Immunohistochemical examination stained positive for alpha smooth muscle actin, collagen type I, and fibronectin. There was minor tissue formation in the nonconditioned control samples. Porous P4HB may be used to fabricate a biodegradable patch scaffold. Human vascular cells attached themselves to the polymeric scaffold, and extracellular matrix formation was induced under controlled biomechanical and biodynamic stimuli in a self-developed pulsatile bioreactor system.

Effects of feeding condensed distillers solubles and crude glycerin alone or in combination on finishing beef cattle performance, carcass characteristics, and in vitro fermentation.

USDA-ARS?s Scientific Manuscript database

Two experiments were conducted to evaluate the effects of feeding condensed distillers solubles (DS) and crude glycerin alone or in combination on performance of finishing beef cattle and in vitro fermentation. In both experiments, dietary treatments consisted of a steam flaked corn (SFC) based diet...

Stryphnodendron adstringens and purified tannin on Pythium insidiosum: in vitro and in vivo studies.

PubMed

Trolezi, Rodrigo; Azanha, Juliana Maziero; Paschoal, Natália Rodrigues; Chechi, Jéssica Luana; Dias Silva, Marcelo José; Fabris, Viciany Eric; Vilegas, Wagner; Kaneno, Ramon; Fernandes Junior, Ary; Bosco, Sandra de Moraes Gimenes

2017-02-23

Pythium insidiosum is the etiological agent of pythiosis, an emerging life-threatening infectious disease in tropical and subtropical regions. The pathogen is a fungus-like organism resistant to antifungal therapy, for this reason, most cases need extensive surgical debridments as treatment, but depending on the size and anatomical region of the lesion, such approach is unfeasible. We investigate the fungicidal effect and toxicity of crude bark extract of Stryphnodendron adstringens and commercially available tannin on Pythium insidiosum both in vitro and in vivo. Standardized fragments of mycelia of fifteen isolates of P. insidiosum were tested with different concentrations of bark extract (10 to 30% v/v) and tannin (0.5, 1.0 and 1.5 mg/mL). For in vivo study, fifteen rabbits were experimentally infected with zoospores of P. insidiosum and treated by oral and intralesional applications of bark extract and tannin. Acute toxicity tests with both substances were also performed in rats. In vitro studies showed fungicidal effect for both substances at different concentrations and the SEM showed alteration on the cell wall surface of the pathogen. All infected rabbits developed a firm nodular mass that reached around 90 mm 2 ninety days after inoculation, but neither the intralesional inoculation of tannin, nor the oral administration of crude extract and tannin were able to promote remission of the lesions. Lesions developed by rabbits presented an encapsulated abscess being quite different of naturally acquired pythiosis, which is characterized by ulcerated lesions. Since no toxicity was observed in rats or rabbits inoculated with these products, while in vitro experiments showed direct antifungal effect, therapeutic activity of S. adstringens and tannin should be clinically tested as an alternative for healing wounds in naturally acquired pythiosis.

Model-Based Analysis of Biopharmaceutic Experiments To Improve Mechanistic Oral Absorption Modeling: An Integrated in Vitro in Vivo Extrapolation Perspective Using Ketoconazole as a Model Drug.

PubMed

Pathak, Shriram M; Ruff, Aaron; Kostewicz, Edmund S; Patel, Nikunjkumar; Turner, David B; Jamei, Masoud

2017-12-04

Mechanistic modeling of in vitro data generated from metabolic enzyme systems (viz., liver microsomes, hepatocytes, rCYP enzymes, etc.) facilitates in vitro-in vivo extrapolation (IVIV_E) of metabolic clearance which plays a key role in the successful prediction of clearance in vivo within physiologically-based pharmacokinetic (PBPK) modeling. A similar concept can be applied to solubility and dissolution experiments whereby mechanistic modeling can be used to estimate intrinsic parameters required for mechanistic oral absorption simulation in vivo. However, this approach has not widely been applied within an integrated workflow. We present a stepwise modeling approach where relevant biopharmaceutics parameters for ketoconazole (KTZ) are determined and/or confirmed from the modeling of in vitro experiments before being directly used within a PBPK model. Modeling was applied to various in vitro experiments, namely: (a) aqueous solubility profiles to determine intrinsic solubility, salt limiting solubility factors and to verify pK a ; (b) biorelevant solubility measurements to estimate bile-micelle partition coefficients; (c) fasted state simulated gastric fluid (FaSSGF) dissolution for formulation disintegration profiling; and (d) transfer experiments to estimate supersaturation and precipitation parameters. These parameters were then used within a PBPK model to predict the dissolved and total (i.e., including the precipitated fraction) concentrations of KTZ in the duodenum of a virtual population and compared against observed clinical data. The developed model well characterized the intraluminal dissolution, supersaturation, and precipitation behavior of KTZ. The mean simulated AUC 0-t of the total and dissolved concentrations of KTZ were comparable to (within 2-fold of) the corresponding observed profile. Moreover, the developed PBPK model of KTZ successfully described the impact of supersaturation and precipitation on the systemic plasma concentration profiles of KTZ for 200, 300, and 400 mg doses. These results demonstrate that IVIV_E applied to biopharmaceutical experiments can be used to understand and build confidence in the quality of the input parameters and mechanistic models used for mechanistic oral absorption simulations in vivo, thereby improving the prediction performance of PBPK models. Moreover, this approach can inform the selection and design of in vitro experiments, potentially eliminating redundant experiments and thus helping to reduce the cost and time of drug product development.

In vitro and in vivo studies of the combination of IGF1R inhibitor figitumumab (CP-751,871) with HER2 inhibitors trastuzumab and neratinib.

PubMed

Chakraborty, Ashok K; Zerillo, Cynthia; DiGiovanna, Michael P

2015-08-01

The insulin-like growth factor I receptor (IGF1R) has been linked to resistance to HER2-directed therapy with trastuzumab (Herceptin). We examined the anti-tumor activity of figitumumab (CP-751,871), a human monoclonal antibody that blocks IGF1R ligand binding, alone and in combination with the therapeutic anti-HER2 antibody trastuzumab and the pan-HER family tyrosine kinase inhibitor neratinib, using in vitro and in vivo breast cancer model systems. In vitro assays of proliferation, apoptosis, and signaling, and in vivo anti-tumor experiments were conducted in HER2-overexpressing (BT474) and HER2-normal (MCF7) models. We find single-agent activity of the HER2-targeting drugs but not figitumumab in the BT474 model, while the reverse is true in the MCF7 model. However, in both models, combining figitumumab with HER2-targeting drugs shows synergistic anti-proliferative and apoptosis-inducing effects, and optimum inhibition of downstream signaling. In murine xenograft models, synergistic anti-tumor effects were observed in the HER2-normal MCF7 model for the combination of figitumumab with trastuzumab, and, in the HER2-overexpressing BT474 model, enhanced anti-tumor effects were observed for the combination of figitumumab with either trastuzumab or neratinib. Analysis of tumor extracts from the in vivo experiments showed evidence of the most optimal inhibition of downstream signaling for the drug combinations over the single-agent therapies. These results suggest promise for such combinations in treating patients with breast cancer, and that, unlike the case for single-agent therapy, the therapeutic effects of such combinations may be independent of expression levels of the individual receptors or the single-agent activity profile.

A Second Las17 Monomeric Actin-Binding Motif Functions in Arp2/3-Dependent Actin Polymerization During Endocytosis

PubMed Central

Feliciano, Daniel; Tolsma, Thomas O.; Farrell, Kristen B.; Aradi, Al; Di Pietro, Santiago M.

2018-01-01

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME. PMID:25615019

Study on fluorouracil-chitosan nanoparticle preparation and its antitumor effect.

PubMed

Chen, Gaimin; Gong, Rudong

2016-05-01

To successfully prepare fluorouracil-chitosan nanoparticles, and further analyze its anti-tumor activity mechanism, this paper makes a comprehensive study of existing preparation prescription and makes a detailed analysis of fluorouracil-chitosan in vitro release and pharmacodynamic behavior of animals. Two-step synthesis method is adopted to prepare 5-FU-CS-mPEG prodrugs, and infrared, (1)H NMR and differential thermal analysis are adopted to analyze characterization synthetic products of prepared drugs. To ensure clinical efficacy of prepared drugs, UV spectrophotometry is adopted for determination of drug loading capacity of prepared drugs, transmission electron microscopy is adopted to observe the appearance, dynamic dialysis method is used to observe in vitro drug release of prepared drugs and fitting of various release models is done. Anti-tumor effect is studied via level of animal pharmacodynamics. After the end of the experiment, tumor inhibition rate, spleen index and thymus index of drugs are calculated. Experimental results show that the prepared drugs are qualified in terms of regular shape, dispersion, drug content, etc. Animal pharmacodynamics experiments have shown that concentration level of drug loading capacity of prepared drugs has a direct impact on anti-tumor rate. The higher the concentration, the higher the anti-tumor rate. Results of pathological tissue sections of mice show that the prepared drugs cause varying degrees of damage to receptor cells, resulting in cell necrosis or apoptosis problem. It can thus be concluded that ion gel method is an effective method to prepare drug-loading nanoparticles, with prepared nanoparticles evenly distributed in regular shape which demonstrate good slow-release characteristics in receptor vitro and vivo. At the same time, after completion of drug preparation, relatively strong anti-tumor activity can be generated for the receptor, so this mode of preparation enjoys broad prospects for development.

Presence of nano-sized silica during in vitro digestion of foods containing silica as a food additive.

PubMed

Peters, Ruud; Kramer, Evelien; Oomen, Agnes G; Rivera, Zahira E Herrera; Oegema, Gerlof; Tromp, Peter C; Fokkink, Remco; Rietveld, Anton; Marvin, Hans J P; Weigel, Stefan; Peijnenburg, Ad A C M; Bouwmeester, Hans

2012-03-27

The presence, dissolution, agglomeration state, and release of materials in the nano-size range from food containing engineered nanoparticles during human digestion is a key question for the safety assessment of these materials. We used an in vitro model to mimic the human digestion. Food products subjected to in vitro digestion included (i) hot water, (ii) coffee with powdered creamer, (iii) instant soup, and (iv) pancake which either contained silica as the food additive E551, or to which a form of synthetic amorphous silica or 32 nm SiO(2) particles were added. The results showed that, in the mouth stage of the digestion, nano-sized silica particles with a size range of 5-50 and 50-500 nm were present in food products containing E551 or added synthetic amorphous silica. However, during the successive gastric digestion stage, this nano-sized silica was no longer present for the food matrices coffee and instant soup, while low amounts were found for pancakes. Additional experiments showed that the absence of nano-sized silica in the gastric stage can be contributed to an effect of low pH combined with high electrolyte concentrations in the gastric digestion stage. Large silica agglomerates are formed under these conditions as determined by DLS and SEM experiments and explained theoretically by the extended DLVO theory. Importantly, in the subsequent intestinal digestion stage, the nano-sized silica particles reappeared again, even in amounts higher than in the saliva (mouth) digestion stage. These findings suggest that, upon consumption of foods containing E551, the gut epithelium is most likely exposed to nano-sized silica. © 2012 American Chemical Society

Alleviation of streptozotocin-induced diabetes in nude mice by stem cells derived from human first trimester umbilical cord.

PubMed

Cao, M; Zhang, J B; Dong, D D; Mou, Y; Li, K; Fang, J; Wang, Z Y; Chen, C; Zhao, J; Yie, S M

2015-10-16

Cells isolated from human first trimester umbilical cord perivascular layer (hFTM-PV) tissues display the pluripotent characteristics of stem cells. In this study, we examined whether hFTM-PV cells can differentiate into islet-like clusters (ILCs) in vitro, and whether transplantation of the hFTM-PV cells with and without differentiation in vitro can alleviate diabetes in nude mice. The hFTM-PV cells were differentiated into ILCs in vitro through a simple stepwise culture protocol. To examine the in vivo effects of the cells, the hFTM-PV cells with and without differentiation in vitro were transplanted into the abdominal cavity of nude mice with streptozotocin (STZ)-induced diabetes. Blood glucose levels, body weight, and the survival probability of the diabetic nude mice were then statistically analyzed. The hFTM-PV cells were successfully induced into ILCs that could release insulin in response to elevated concentrations of glucose in vitro. In transplantation experiments, we observed that mice transplanted with the undifferentiated hFTM-PV cells, embryonic body-like cell aggregations, or ILCs all demonstrated normalized hyperglycemia and showed improved survival rate compared with those without cell transplantation. The hFTM-PV cells have the ability to differentiate into ILCs in vitro and transplantations of undifferentiated and differentiated cells can alleviate STZ-induced diabetes in nude mice. This may offer a potential cell source for stem cell-based therapy for treating diabetes in the future.

Diffusion of Antimicrobials Across Silicone Hydrogel Contact Lenses

PubMed Central

Zambelli, Alison M.; Brothers, Kimberly M.; Hunt, Kristin M.; Romanowski, Eric G.; Nau, Amy C.; Dhaliwal, Deepinder K.; Shanks, Robert M. Q.

2014-01-01

Objectives To measure the diffusion of topical preparations of moxifloxacin, amphotericin B (AmB), and polyhexamethylene biguanide (PHMB) through silicone hydrogel (SH) contact lenses in vitro. Methods Using an in vitro model, the diffusion of three antimicrobials through SH contact lenses was measured. Diffused compounds were measured using a spectrophotometer at set time points over a period of four hours. The amount of each diffused antimicrobial was determined by comparing the experimental value to a standard curve. A biological assay was performed to validate the contact lens diffusion assay by testing antimicrobial activity of diffused material against lawns of susceptible bacteria (Staphylococcus epidermidis) and yeast (Saccharomyces cerevisiae). Experiments were repeated at least two times with a total of at least 4 independent replicates. Results Our data show detectable moxifloxacin and PHMB diffusion through SH contact lenses at 30 minutes, while amphotericin B diffusion remained below the limit of detection within the 4 hour experimental period. In the biological assay, diffused moxifloxacin demonstrated microbial killing starting at 20 minutes on bacterial lawns, whereas PHMB and amphotericin B failed to demonstrate killing on microbial lawns over the course of the 60 minute experiment. Conclusions In vitro diffusion assays demonstrate limited penetration of certain anti-infective agents through silicone hydrogel contact lenses. Further studies regarding the clinical benefit of using these agents along with bandage contact lens use for corneal pathology are warranted. PMID:25806673

The Genome-Wide Expression Profile of Saussurea lappa Extract on House Dust Mite-Induced Atopic Dermatitis in Nc/Nga Mice.

PubMed

Lim, Hye-Sun; Ha, Hyekyung; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

2015-09-01

Saussurea lappa has been reported to possess anti-atopic properties. In this study, we have confirmed the S. lappa's anti-atopic properties in Nc/Nga mice and investigated the candidate gene related with its properties using microarray. We determined the target gene using real time PCR in in vitro experiment. S. lappa showed the significant reduction in atopic dermatitis (AD) score and immunoglobulin E compared with the AD induced Nc/Nga mice. In the results of microarray using back skin obtained from animals, we found that S. lappa's properties are closely associated with cytokine-cytokine receptor interaction and the JAK-STAT signaling pathway. Consistent with the microarray data, real-time RT-PCR confirmed these modulation at the mRNA level in skin tissues from S. lappa-treated mice. Among these genes, PI3Kca and IL20Rβ were significantly downregulated by S. lappa treatment in Nc/Nga mouse model. In in vitro experiment using HaCaT cells, we found that the S. lappa components, including alantolactone, caryophyllene, costic acid, costunolide and dehydrocostus lactone significantly decreased the expression of PI3Kca but not IL20Rβ in vitro. Therefore, our study suggests that PI3Kca-related signaling is closely related with the protective effects of S. lappa against the development of atopic-dermatitis.

Characterization and In Vitro and In Vivo Assessment of a Novel Cellulose Acetate-Coated Mg-Based Alloy for Orthopedic Applications

PubMed Central

Neacsu, Patricia; Staras, Adela Ioana; Voicu, Stefan Ioan; Ionascu, Iuliana; Soare, Teodoru; Uzun, Seralp; Cojocaru, Vasile Danut; Pandele, Andreea Madalina; Croitoru, Sorin Mihai; Miculescu, Florin; Cotrut, Cosmin Mihai; Dan, Ioan; Cimpean, Anisoara

2017-01-01

Despite their good biocompatibility and adequate mechanical behavior, the main limitation of Mg alloys might be their high degradation rates in a physiological environment. In this study, a novel Mg-based alloy exhibiting an elastic modulus E = 42 GPa, Mg-1Ca-0.2Mn-0.6Zr, was synthesized and thermo-mechanically processed. In order to improve its performance as a temporary bone implant, a coating based on cellulose acetate (CA) was realized by using the dipping method. The formation of the polymer coating was demonstrated by FT-IR, XPS, SEM and corrosion behavior comparative analyses of both uncoated and CA-coated alloys. The potentiodynamic polarization test revealed that the CA coating significantly improved the corrosion resistance of the Mg alloy. Using a series of in vitro and in vivo experiments, the biocompatibility of both groups of biomaterials was assessed. In vitro experiments demonstrated that the media containing their extracts showed good cytocompatibility on MC3T3-E1 pre-osteoblasts in terms of cell adhesion and spreading, viability, proliferation and osteogenic differentiation. In vivo studies conducted in rats revealed that the intramedullary coated implant for fixation of femur fracture was more efficient in inducing bone regeneration than the uncoated one. In this manner, the present study suggests that the CA-coated Mg-based alloy holds promise for orthopedic aplications. PMID:28773046

The MTA family proteins as novel histone H3 binding proteins.

PubMed

Wu, Meng; Wang, Lina; Li, Qian; Li, Jiwen; Qin, Jun; Wong, Jiemin

2013-01-03

The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

The MTA family proteins as novel histone H3 binding proteins

PubMed Central

2013-01-01

Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail. PMID:23286669

Effects of heavy metals on ultrastructure and Hsp70 induction in Lemna minor L. exposed to water along the Sarno River, Italy.

PubMed

Basile, A; Sorbo, S; Cardi, M; Lentini, M; Castiglia, D; Cianciullo, P; Conte, B; Loppi, S; Esposito, S

2015-04-01

The effects of freshwater pollution in the highly contaminated river Sarno (Campania, Southern Italy) have been evaluated using bags containing the aquatic plant Lemna minor (Lemnacee, Arales), in order to determine morpho-physiological modifications as a response to pollutants. The exposition of Lemna bags for 7 days on three different sites along the river path showed alterations in chloroplasts and vacuoles shape and organization. Moreover, some specimens were exposed in vitro at the same heavy metal (HM) concentrations measured in the polluted sites of the river, and compared with data from the bag experiment; to verify the dose and time dependent effects, samples were exposed to HM in vitro at concentrations ranging from 10(-6) to 10(-4)M up to 7 days. Transmission electron microscopy (TEM) observations on in vitro plants confirmed that ultrastructural alterations affected most of plastids and the shape of different subcellular structures, namely vacuoles; in in vitro stressed specimens, Heat Shock Proteins 70 (Hsp70) levels changed, in dependence of changing levels of HM measured in different sites along the river path. Thus L. minor exhibited a possible correlation between the levels of HM pollution and Hsp70 occurrence; interestingly, the data presented showed that copper specifically increased Hsp70 levels at concentrations detected in polluted river waters, whereas cadmium and lead did not; on the other side, the latter represent highly toxic elements when specimens were exposed to higher levels in vitro. The effects of specific elements in vitro are compared to those observed in bags exposed along the river path; thus results are examined in order to propose L. minor as an organism able to be utilized to monitor heavy metals pollution; the possibility of using Hsp70s as specific markers of HM pollution is discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs.

PubMed

Park, Kyu Ree; Park, Chan Sol; Kim, Beob Gyun

2016-01-01

Two experiments were conducted to determine the effects of enzyme complex on in vitro dry matter (DM) digestibility for feed ingredients. The objective of experiment 1 was to screen feed ingredients that can be effective substrates for an enzyme complex, mainly consisted of β-pentosanase, β-glucanase and α-amylase, using in vitro digestibility methods. In experiment 1, the test ingredients were three grain sources (barley, corn and wheat) and six protein supplements (canola meal, copra expellers, cottonseed meal, distillers dried grains with solubles, palm kernel expellers and soybean meal). In vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM for test ingredients were determined. In vitro digestibility methods consisted of two- or three-step procedure simulating in vivo digestion in the pig gastrointestinal tracts with or without enzyme complex. As the enzyme complex added, the IVID of DM for corn and wheat increased (p < 0.05) by 5.0 and 2.6 percentage unit, respectively. The IVTTD of DM for corn increased (p < 0.05) by 3.1 percentage unit with enzyme complex addition. As the effect of enzyme complex was the greatest in corn digestibility, corn grains were selected to determine the in vitro digestibility of the fractions (starch, germ, hull and gluten) that maximally respond to the enzyme complex in experiment 2. The IVID of DM for corn starch, germ and hull increased (p < 0.05) by 16.0, 2.8 and 1.2 percentage unit, respectively. The IVTTD of DM for corn starch and hull also increased (p < 0.05) by 8.6 and 0.9 percentage unit, respectively, with enzyme complex addition. In conclusion, the enzyme complex increases in vitro DM digestibility of corn and wheat, and the digestibility increments of corn are mainly attributed to the increased digestibility of corn starch.

Distribution of Triamcinolone Acetonide after Intravitreal Injection into Silicone Oil-Filled Eye.

PubMed

Da, Ma; Li, Kenneth K W; Chan, Kevin C; Wu, Ed X; Wong, David S H

2016-01-01

There is increasing use of the vitreous cavity as a reservoir for drug delivery. We study the intraocular migration and distribution of triamcinolone acetonide (TA) after injection into silicone oil tamponade agent during and after vitrectomy surgery ex vivo (pig eye) and in vitro (glass bottle). For ex vivo assessment, intraocular migration of TA was imaged using real-time FLASH MRI scans and high-resolution T2W imaging and the in vitro model was monitored continuously with a video camera. Results of the ex vivo experiment showed that the TA droplet sank to the interface of silicone oil and aqueous almost immediately after injection and remained inside the silicone oil bubble for as long as 16 minutes. The in vitro results showed that, after the shrinkage of the droplet, TA gradually precipitated leaving only a lump of whitish crystalline residue inside the droplet for about 100 minutes. TA then quickly broke the interface and dispersed into the underlying aqueous within 15 seconds, which may result in a momentary increase of local TA concentration in the aqueous portion and potentially toxic to the retina. Our study suggests that silicone oil may not be a good candidate as a drug reservoir for drugs like TA.

A tetracycline expression system in combination with Sox9 for cartilage tissue engineering.

PubMed

Yao, Yi; He, Yu; Guan, Qian; Wu, Qiong

2014-02-01

Cartilage tissue engineering using controllable transcriptional therapy together with synthetic biopolymer scaffolds shows higher potential for overcoming chondrocyte degradation and constructing artificial cartilages both in vivo and in vitro. Here, the potential regulating tetracycline expression (Tet-on) system was used to express Sox9 both in vivo and in vitro. Chondrocyte degradation was measured in vitro and overcome by Soxf9 expression. Experiments confirmed the feasibility of the combined use of Sox9 and Tet-on system in cartilage tissue engineering. Engineered poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds were seeded with recombinant chondrocytes which were transfected with Tet-induced Sox9 expression; the scaffolds were implanted under the skin of 8-week-old rats. The experimental group was injected with Dox in the abdomen, while the control group was injected with normal saline. After 4 or 8 days of implantation in vivo, the newly formed pieces of articular chondrocytes were taken out and measured. Dox injection in vivo showed positive effect on recombinant chondrocytes, in which Sox9 expression was up-regulated by an inducible system with specific matrix proteins. The results demonstrate this controllable transcriptional therapy is a potential approach for tissue engineering. Copyright © 2013 Elsevier Ltd. All rights reserved.

Formulation and in vitro/in vivo evaluation of levodopa transdermal delivery systems.

PubMed

Lee, Kyung Eun; Choi, Yun Jung; Oh, Byu Ree; Chun, In Koo; Gwak, Hye Sun

2013-11-18

This study aims to investigate the feasibility of Levodopa transdermal delivery systems (TDSs). Levodopa TDSs were formulated using various vehicles and permeation enhancers, and in vitro permeation and in vivo pharmacokinetic studies were carried out. In the in vitro study, ester-type vehicles showed relatively high enhancing effects; propylene glycol monocaprylate and propylene glycol monolaurate showed the highest permeation fluxes from both solution and pressure sensitive adhesive (PSA) TDS formulations. Lag time was dramatically shortened with PSA TDS formulations as compared with solution formulations. In the in vivo study, the addition of fatty acids increased blood drug concentrations regardless of the kind or concentration of fatty acid; the AUCinf increased up to 8.7 times as compared with propylene glycol (PG) alone. PSA TDS containing 10% linoleic acid exhibited prolonged Tmax as compared with oral form. Total clearance of L-dopa from PSA TDSs was significantly lower than from oral form (up to 86.8 times). Especially, PSA TDS containing 10% linoleic acid (LOA) revealed 76.2 fold higher AUCinf than oral administration. Based on our results, the L-dopa PSA TDS containing PG with 10% LOA could be used as a good adjuvant therapy for Parkinson's disease patients who experience symptom fluctuation by L-dopa oral administration. Copyright © 2013 Elsevier B.V. All rights reserved.

In vitro and in vivo characterisation of PEG-lipid-based micellar complexes of salmon calcitonin for pulmonary delivery.

PubMed

Baginski, Leonie; Gobbo, Oliviero L; Tewes, Frederic; Salomon, Johanna J; Healy, Anne Marie; Bakowsky, Udo; Ehrhardt, Carsten

2012-06-01

To investigate DSPE-PEG(2000)-based micellar formulations of salmon calcitonin (sCT) for their ability to improve pulmonary delivery. Micelles were characterised by DLS and (31)P-NMR spectroscopy. Stability against sCT degrading peptidases, trypsin, α-chymotrypsin and neutrophil elastase as well as their influence on transepithelial absorption was investigated in vitro. In vivo performance of sCT micelles was studied in an experimental model of intratracheal aerosolisation into rats. Micelles with a mean hydrodynamic diameter of 12 nm spontaneously assembled, when a total concentration of 0.02 mM of PEG-lipid and sCT (at 1:1 molar ratio) was exceeded. Nuclear magnetic resonance confirmed the presence of small micellar structures. The micellar formulation showed increased stability against enzymatic digestion. In vitro studies also showed that sCT micelles were able to enhance transepithelial absorption. Data obtained from in vivo experiments provided evidence of significantly (P < 0.05) higher mean plasma concentrations of sCT, after inhalation of micelles compared to sCT solution, at 60 and 90 min, a significantly higher AUC (inf) and a relative bioavailability of 160 ± 55% when compared to plain sCT solution. The herein described PEG-lipid micelles are promising carriers for enhanced pulmonary delivery of sCT.

Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding.

PubMed

Tenzer, S; Peters, B; Bulik, S; Schoor, O; Lemmel, C; Schatz, M M; Kloetzel, P-M; Rammensee, H-G; Schild, H; Holzhütter, H-G

2005-05-01

Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).

The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles

PubMed Central

Cho, Eun Chul; Zhang, Qiang; Xia, Younan

2011-01-01

In vitro experiments typically measure the uptake of nanoparticles by exposing cells at the bottom of a culture plate to a suspension of nanoparticles, which is assumed to be well-dispersed. However, nanoparticles can sediment and this means the concentration of particles on the cell surface and those actually taken up by the cells may be higher than the initial bulk concentration. Here we use upright and inverted cell culture configurations to show that cellular uptake of gold nanoparticles depends on the sedimentation and diffusion velocities of the nanoparticles and is independent of size, shape, density, surface coating and initial concentration of the nanoparticles. Generally more nanoparticles are taken up in the upright configuration than the inverted one and nanoparticles that sediment faster showed greater differences in uptake between the two configurations. Our results suggest that cellular uptake of nanoparticles is sensitive to the way cells are positioned and sedimentation need to be considered when performing in vitro studies for large and heavy nanoparticles. PMID:21516092

Inflammatory response study of gellan gum impregnated duck's feet derived collagen sponges.

PubMed

Song, Jeong Eun; Lee, Seon Eui; Cha, Se Rom; Jang, Na Keum; Tripathy, Nirmalya; Reis, Rui L; Khang, Gilson

2016-10-01

Tissue engineered biomaterials have biodegradable and biocompatible properties. In this study, we have fabricated sponges using duck's feet derived collagen (DC) and gellan gum (GG), and further studied its inflammatory responses. The as-prepared duck's feet DC/GG sponges showed the possibility of application as a tissue engineering material through in vitro and in vivo experiments. The physical and chemical properties of sponges were characterized by compression strength, porosity, and scanning electron microscopy, etc. In vitro cell viability were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. An inflammatory response was studied after seeding RAW264.7 cells on as-fabricated sponges using reverse transcriptase-polymerase chain reaction. In vivo studies were carried out by implanting in subcutaneous nude mouse followed by extraction, histological staining. Collectively, superior results were showed by DC/GG sponges than GG sponge in terms of physical property and cell proliferation and thus can be considered as a potential candidate for future tissue engineering applications.

Computational analysis of blood clot dissolution using a vibrating catheter tip.

PubMed

Lee, Jeong Hyun; Oh, Jin Sun; Yoon, Bye Ri; Choi, Seung Hong; Rhee, Kyehan; Jho, Jae Young; Han, Moon Hee

2012-04-01

We developed a novel concept of endovascular thrombolysis that employs a vibrating electroactive polymer actuator. In order to predict the efficacy of thrombolysis using the developed vibrating actuator, enzyme (plasminogen activator) perfusion into a clot was analyzed by solving flow fields and species transport equations considering the fluid structure interaction. In vitro thrombolysis experiments were also performed. Computational results showed that plasminogen activator perfusion into a clot was enhanced by actuator vibration at frequencies of 1 and 5 Hz. Plasminogen activator perfusion was affected by the actuator oscillation frequencies and amplitudes that were determined by electromechanical characteristics of a polymer actuator. Computed plasminogen activator perfused volumes were compared with experimentally measured dissolved clot volumes. The computed plasminogen activator perfusion volumes with threshold concentrations of 16% of the initial plasminogen activator concentration agreed well with the in vitro experimental data. This study showed the effectiveness of actuator oscillation on thrombolysis and the validity of the computational plasminogen activator perfusion model for predicting thrombolysis in complex flow fields induced by an oscillating actuator.

Identification of ACE-inhibitory peptides from Phaseolus vulgaris after in vitro gastrointestinal digestion.

PubMed

Tagliazucchi, Davide; Martini, Serena; Bellesia, Andrea; Conte, Angela

2015-01-01

The objective of this study was to identify the angiotensin I-converting enzyme (ACE)-inhibitory peptides released from thermally treated Phaseolus vulgaris (pinto) whole beans after in vitro gastrointestinal digestion. The degree of hydrolysis increased during digestion reaching a value of 50% at the end of the pancreatic digestion. The <3 kDa fraction of the postpancreatic sample showed high ACE-inhibitory activity (IC50 = 105.6 ± 2.1 μg of peptides/mL). Peptides responsible for the ACE-inhibitory activity were isolated by reverse-phase high-performance liquid chromatography (HPLC). Three fractions, showing the highest inhibitory activity, were selected for tandem mass spectrometry (MS/MS) experiments. Eleven of the identified sequences have previously been described as ACE-inhibitors. Most of the identified bioactive peptides have a hydrophobic amino acid, (iso)leucine or phenylalanine, or proline at the C-terminal position, which is crucial for their ACE-inhibitory activity. The sequence of some peptides allowed us to anticipate the presence of ACE-inhibitory activity.

Macrogenomic engineering via modulation of the scaling of chromatin packing density.

PubMed

Almassalha, Luay M; Bauer, Greta M; Wu, Wenli; Cherkezyan, Lusik; Zhang, Di; Kendra, Alexis; Gladstein, Scott; Chandler, John E; VanDerway, David; Seagle, Brandon-Luke L; Ugolkov, Andrey; Billadeau, Daniel D; O'Halloran, Thomas V; Mazar, Andrew P; Roy, Hemant K; Szleifer, Igal; Shahabi, Shohreh; Backman, Vadim

2017-11-01

Many human diseases result from the dysregulation of the complex interactions between tens to thousands of genes. However, approaches for the transcriptional modulation of many genes simultaneously in a predictive manner are lacking. Here, through the combination of simulations, systems modelling and in vitro experiments, we provide a physical regulatory framework based on chromatin packing-density heterogeneity for modulating the genomic information space. Because transcriptional interactions are essentially chemical reactions, they depend largely on the local physical nanoenvironment. We show that the regulation of the chromatin nanoenvironment allows for the predictable modulation of global patterns in gene expression. In particular, we show that the rational modulation of chromatin density fluctuations can lead to a decrease in global transcriptional activity and intercellular transcriptional heterogeneity in cancer cells during chemotherapeutic responses to achieve near-complete cancer cell killing in vitro. Our findings represent a 'macrogenomic engineering' approach to modulating the physical structure of chromatin for whole-scale transcriptional modulation.

Identification of the anti-tumor activity and mechanisms of nuciferine through a network pharmacology approach

PubMed Central

Qi, Quan; Li, Rui; Li, Hui-ying; Cao, Yu-bing; Bai, Ming; Fan, Xiao-jing; Wang, Shu-yan; Zhang, Bo; Li, Shao

2016-01-01

Aim: Nuciferine is an aporphine alkaloid extracted from lotus leaves, which is a raw material in Chinese medicinal herb for weight loss. In this study we used a network pharmacology approach to identify the anti-tumor activity of nuciferine and the underlying mechanisms. Methods: The pharmacological activities and mechanisms of nuciferine were identified through target profile prediction, clustering analysis and functional enrichment analysis using our traditional Chinese medicine (TCM) network pharmacology platform. The anti-tumor activity of nuciferine was validated by in vitro and in vivo experiments. The anti-tumor mechanisms of nuciferine were predicted through network target analysis and verified by in vitro experiments. Results: The nuciferine target profile was enriched with signaling pathways and biological functions, including “regulation of lipase activity”, “response to nicotine” and “regulation of cell proliferation”. Target profile clustering results suggested that nuciferine to exert anti-tumor effect. In experimental validation, nuciferine (0.8 mg/mL) markedly inhibited the viability of human neuroblastoma SY5Y cells and mouse colorectal cancer CT26 cells in vitro, and nuciferine (0.05 mg/mL) significantly suppressed the invasion of 6 cancer cell lines in vitro. Intraperitoneal injection of nuciferine (9.5 mg/mL, ip, 3 times a week for 3 weeks) significantly decreased the weight of SY5Y and CT26 tumor xenografts in nude mice. Network target analysis and experimental validation in SY5Y and CT26 cells showed that the anti-tumor effect of nuciferine was mediated through inhibiting the PI3K-AKT signaling pathway and IL-1 levels in SY5Y and CT26 cells. Conclusion: By using a TCM network pharmacology method, nuciferine is identified as an anti-tumor agent against human neuroblastoma and mouse colorectal cancer in vitro and in vivo, through inhibiting the PI3K-AKT signaling pathways and IL-1 levels. PMID:27180984

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish.

PubMed

Segner, Helmut

2004-10-01

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.

In vitro percutaneous absorption studies and in vivo evaluation of anti-inflammatory activity of essential fatty acids (EFA) from fish oil extracts.

PubMed

Puglia, Carmelo; Tropea, Salvatore; Rizza, Luisa; Santagati, Natale Alfredo; Bonina, Francesco

2005-08-11

The aim of the present study was to evaluate the in vitro percutaneous absorption and the in vivo anti-inflammatory activity of EPA and DHA fatty acids from three oily extracts, obtained by acetonic extractions from the entrails of different varieties of Mediterranean fishes such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus). In the first part of our research, we focused our attention on the characterization of the oily extracts to determine their omega-3 polyunsaturated fatty acid content, then, we evaluated the in vitro percutaneous absorption through excised human skin (stratum corneum/epidermis membranes; SCE) of EPA and DHA contained in the extracts. In the second part, the fish oil which guaranteed the best in vitro permeation profile of these omega-3 fatty acids was studied in order to evaluate its inhibiting ability towards the in vivo UVB-induced skin erythema. From the results obtained, all the fish oils tested in this study presented significant amounts of omega-3 fatty acids EPA and DHA, and particularly sardine oil extract showed higher concentrations of these substances compared to the other two fish oils. The in vitro experiments revealed interesting fluxes of these compounds from sardine extract through the stratum corneum/epidermis membranes and an appreciable anti-inflammatory activity against UVB-induced erythema in human volunteers was also observed.

Hydroxyapatite nanorods: soft-template synthesis, characterization and preliminary in vitro tests.

PubMed

Nguyen, Nga Kim; Leoni, Matteo; Maniglio, Devid; Migliaresi, Claudio

2013-07-01

Synthetic hydroxyapatite nanorods are excellent candidates for bone tissue engineering applications. In this study, hydroxyapatite nanorods resembling bone minerals were produced by using soft-template method with cetyltrimethylammonium bromide. Composite hydroxyapatite/poly(D, L)lactic acid films were prepared to evaluate the prepared hydroxyapatite nanorods in terms of cell affinity. Preliminary in vitro experiments showed that aspect ratio and film surface roughness play a vital role in controlling adhesion and proliferation of human osteoblast cell line MG 63. The hydroxyapatite nanorods with aspect ratios in the range of 5.94-7 were found to possess distinctive properties, with the corresponding hydroxyapatite/poly(D, L)lactic acid films promoting cellular confluence and a fast formation of collagen fibers as early as after 7 days of culture.

In vitro simulation studies of silica deposition induced by lignin from rice*

PubMed Central

Fang, Jiang-Yu; Ma, Xue-Long

2006-01-01

To reveal the possible mechanism of silica deposition in higher plants, lignin was isolated from rice straw following a modified method to conduct a simulation experiment in vitro. UV and infrared absorption spectra showed that the substance had the unique characteristics of pure lignin. The presence of silicon in the precipitation was revealed by TEM (transmission electron microscopy) with EDXA (energy dispersive X-ray analysis) device. It was found that in the borax solution where lignin precipitation occurred silica-lignin co-precipitation was produced but not in the DMSO solution where lignin was broken into its composition compounds and did not precipitate. This means that it is macromolecular lignin itself but not its compounds that could induce silica deposition in higher plants. PMID:16532527

Transdermal permeation of geniposide in the herbal complex liniment in vivo and in vitro.

PubMed

Wang, Yugang; Li, Lele; Li, Huiying; Zhu, Zhaoyun; Hua, Lei; Lei, Fan; Kheir, Michael M; Du, Lijun

2010-06-15

Zhongtong Caji, a kind of liniment, is a traditional Chinese medicinal formula that is widely used for clinical treatment of inflammation and sprains. In this study, the principal effective compound of this formula, geniposide, was used as a criterion to represent the transdermal permeability of the whole formula. A passive diffusion of Zhongtong Caji through the stratum corneum was discovered by an in vitro experiment. The dosage-content relationship detected in subcutaneous tissue after in vivo drug administration was further evidence of its permeation. Blood analysis after different dosages showed that the geniposide could be absorbed and accumulated by subcutaneous tissue within 1h after drug administration, and it would be eliminated by blood circulation 1h after drug treatment. Copyright 2010 Elsevier B.V. All rights reserved.

Influence of Temperature on Transdermal Penetration Enhancing Mechanism of Borneol: A Multi-Scale Study

PubMed Central

Yin, Qianqian; Wang, Ran; Yang, Shufang; Wu, Zhimin; Guo, Shujuan; Dai, Xingxing; Qiao, Yanjiang; Shi, Xinyuan

2017-01-01

The influence of temperature on the transdermal permeation enhancing mechanism of borneol (BO) was investigated using a multi-scale method, containing a coarse-grained molecular dynamic (CG-MD) simulation, an in vitro permeation experiment, and a transmission electron microscope (TEM) study. The results showed that BO has the potential to be used as a transdermal penetration enhancer to help osthole (OST) penetrate into the bilayer. With the increasing temperature, the stratum corneum (SC) becomes more flexible, proving to be synergistic with the permeation enhancement of BO, and the lag time (TLag) of BO and OST are shortened. However, when the temperature increased too much, with the effect of BO, the structure of SC was destroyed; for example, a water pore was formed and the micelle reversed. Though there were a number of drugs coming into the SC, the normal bilayer structure was absent. In addition, through comparing the simulation, in vitro experiment, and TEM study, we concluded that the computer simulation provided some visually detailed information, and the method plays an important role in related studies of permeation. PMID:28106833

In Vitro antifungal activity of essential oils against Colletotrichum gloeosporioides

NASA Astrophysics Data System (ADS)

Yusoff, Nor Hanis Aifaa; Abdullah, Siti Aisyah; Othman, Zaulia; Zainal, Zamri

2018-04-01

The efficacy of Citrus hystrix, Azadirachta indica and Cymbopogon citratus essential oils were evaluated for controlling the growth of mycelia and spore germination of Colletotrichum gloeosporioides. In order to determine the best essential oil (EO) and suitable concentration of essential oil, in vitro experiment was conducted by preparing a pure culture of antrachnose on Potato Dextrose Agar containing EOs of C. hystrix, A. indica and C. citratus with different concentrations (0.2%, 0.6%, 1% and 1.4% (v/v)). The result shows that C. hystrix essential oil at a concentration of 1.4% (v/v) reduced of mycelia growth of C. gloeosporioides by 29.49%. A second experiment was conducted, but at higher concentration of each essential oils (1.8%, 2.2%, 2.6% and 2.8% (v/v)). Significant difference (p ≤ 0.05) inhibition of mycelia growth was obtained in all treatments except the control. The antifungal index values of essential oils were proportionally increased with concentration of essential oil applied in each treatment. It is concluded that essential oil from C. hystrix are efficient in inhibiting C. gloeosporioides.

Defensive properties of pyrrolizidine alkaloids against microorganisms.

PubMed

Joosten, Lotte; van Veen, Johannes A

2011-03-01

The understanding of the selection factors that drive chemical diversification of secondary metabolites of constitutive defence systems in plants, such as pyrrolizidine alkaloids (PAs), is still incomplete. Historically, plants always have been confronted with microorganisms. Long before herbivores existed on this planet, plants had to cope with microbial pathogens. Therefore, plant pathogenic microorganisms may have played an important role in the early evolution of the secondary metabolite diversity. In this review, we discuss the impact that plant-produced PAs have on plant-associated microorganisms. The objective of the review is to present the current knowledge on PAs with respect to anti-microbial activities, adaptation and detoxification by microorganisms, pathogenic fungi, root protection and PA induction. Many in vitro experiments showed effects of PAs on microorganisms. These results point to the potential of microorganisms to be important for the evolution of PAs. However, only a few in vivo studies have been published and support the results of the in vitro studies. In conclusion, the topics pointed out in this review need further exploration by carrying out ecological experiments and field studies.

Effect of seasonal and geographical differences on skin and effect of treatment with an osmoprotectant: Sorbitol.

PubMed

Muizzuddin, Neelam; Ingrassia, Michael; Marenus, Kenneth D; Maes, Daniel H; Mammone, Thomas

2013-01-01

Human skin maintains an optimal permeability barrier function in a terrestrial environment that varies considerably in humidity. Cells cultured under hyperosmotic stress accumulate osmolytes including sorbitol. Epidermal keratinocytes experience similar high osmolality under dry environmental conditions because of increased transepidermal water loss (TEWL) and concomitant drying of the skin. This study was designed to determine if epidermal keratinocytes, in vitro, could be protected from high osmotic stress, with the exogenous addition of sorbitol. In addition, we evaluated the effect of a formulation containing topical sorbitol on skin barrier and moisturization of subjects living in arid and humid regions in summer as well as in winter. Results from in vitro experiments showed that 50 mM sorbitol protected epidermal keratinocytes from osmotic toxicity induced by sodium chloride. Clinical studies indicated that skin chronically exposed to hot, dry environment appeared to exhibit stronger skin barrier and a lower baseline TEWL. In addition, skin barrier was stronger in summer than in winter. Sorbitol exhibited significant improvement in both barrier repair and moisturization, especially in individuals subjected to arid environmental conditions.

Protective effect of arctigenin against MPP+ and MPTP-induced neurotoxicity.

PubMed

Li, Dongwei; Liu, Qingping; Jia, Dong; Dou, Deqiang; Wang, Xiaofei; Kang, Tingguo

2014-01-01

The potential protective effects of arctigenin on 1-methyl-4-phenylpyridinium ion and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyride-induced neurotoxicity were examined, and the results indicated that arctigenin could improve the movement behaviors and upregulate dopamine and γ-aminobutyric acid levels in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyride-induced neurotoxicity mouse model. A further in vitro experiment showed that the pretreatment with arctigenin on cultured human neuroblastoma SH-SY5Y cells could obviously attenuate the decrease of cell survival rates caused by treatment with 1-methyl-4-phenylpyridinium ion by way of acting against cell apoptosis through the decrease of Bax/Bcl-2 and caspase-3, and by antioxidative action through reduction of the surplus reactive oxygen species production and downregulation of mitochondrial membrane potential. It is for the first time that a neuroprotective activity of arctigenin in both in vitro and in vivo experiments was reported, enlightening that arctigenin could be useful as a potential therapeutic agent for Parkinson's disease. Georg Thieme Verlag KG Stuttgart · New York.

A finite element method model to simulate laser interstitial thermo therapy in anatomical inhomogeneous regions

PubMed Central

Mohammed, Yassene; Verhey, Janko F

2005-01-01

Background Laser Interstitial ThermoTherapy (LITT) is a well established surgical method. The use of LITT is so far limited to homogeneous tissues, e.g. the liver. One of the reasons is the limited capability of existing treatment planning models to calculate accurately the damage zone. The treatment planning in inhomogeneous tissues, especially of regions near main vessels, poses still a challenge. In order to extend the application of LITT to a wider range of anatomical regions new simulation methods are needed. The model described with this article enables efficient simulation for predicting damaged tissue as a basis for a future laser-surgical planning system. Previously we described the dependency of the model on geometry. With the presented paper including two video files we focus on the methodological, physical and mathematical background of the model. Methods In contrast to previous simulation attempts, our model is based on finite element method (FEM). We propose the use of LITT, in sensitive areas such as the neck region to treat tumours in lymph node with dimensions of 0.5 cm – 2 cm in diameter near the carotid artery. Our model is based on calculations describing the light distribution using the diffusion approximation of the transport theory; the temperature rise using the bioheat equation, including the effect of microperfusion in tissue to determine the extent of thermal damage; and the dependency of thermal and optical properties on the temperature and the injury. Injury is estimated using a damage integral. To check our model we performed a first in vitro experiment on porcine muscle tissue. Results We performed the derivation of the geometry from 3D ultrasound data and show for this proposed geometry the energy distribution, the heat elevation, and the damage zone. Further on, we perform a comparison with the in-vitro experiment. The calculation shows an error of 5% in the x-axis parallel to the blood vessel. Conclusions The FEM technique proposed can overcome limitations of other methods and enables an efficient simulation for predicting the damage zone induced using LITT. Our calculations show clearly that major vessels would not be damaged. The area/volume of the damaged zone calculated from both simulation and in-vitro experiment fits well and the deviation is small. One of the main reasons for the deviation is the lack of accurate values of the tissue optical properties. In further experiments this needs to be validated. PMID:15631630

Scattering of spermatozoa off cylindrical pillars

NASA Astrophysics Data System (ADS)

Bukatin, Anton; Lushi, Enkeleida; Kantsler, Vasily

2017-11-01

The motion of micro-swimmers in structured environments, even though crucial in processes such as in vivo and in vitro egg fertilization, is still not completely understood. We combine microfluidic experiments with mathematical modeling of 3D swimming near convex surfaces to quantify the dynamics of individual sperm cells in the proximity of cylindrical pillars. Our results show that the hydrodynamic and contact forces that account for the shape asymmetry and flagellar motion, are crucial in correctly describing the dynamics observed in the experiments. Last, we discuss how the size of the cylindrical obstacles determines whether the swimmers scatter off or get trapped circling the pillar.

Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

PubMed

Honkalampi-Hämäläinen, U; Bradley, E L; Castle, L; Severin, I; Dahbi, L; Dahlman, O; Lhuguenot, J-C; Andersson, M A; Hakulinen, P; Hoornstra, D; Mäki-Paakkanen, J; Salkinoja-Salonen, M; Turco, L; Stammati, A; Zucco, F; Weber, A; von Wright, A

2010-03-01

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Contribution of Baicalin on the Plasma Protein Binding Displacement and CYP3A Activity Inhibition to the Pharmacokinetic Changes of Nifedipine in Rats In Vivo and In Vitro

PubMed Central

Gao, Jie; Li, Hong-Meng; Jia, Lin-Jing; Qiao, Hai-Ling

2014-01-01

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), Vd increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism. PMID:24498050

Drug-drug interaction and doping: Effect of non-prohibited drugs on the urinary excretion profile of methandienone.

PubMed

Mazzarino, Monica; Khevenhüller-Metsch, Franziska L; Fiacco, Ilaria; Parr, Maria Kristina; de la Torre, Xavier; Botrè, Francesco

2018-05-15

The potential consequences of drug-drug interactions on the excretion profile of the anabolic androgenic steroid methandienone (17β-hydroxy-17α-methylandrosta-1,4-dien-3-one) are discussed here. More specifically, we have evaluated by in vitro and in vivo experiments the effects of seven non-prohibited drugs (fluconazole, ketoconazole, itraconazole, miconazole, fluoxetine, paroxetine and nefazodone) on the main metabolic pathways of methandienone. These are selected among those most commonly used by the athletes. The in vitro assays were based on the use of human liver microsomes, specific recombinant enzyme isoforms of cytochrome P450 and uridine 5'-diphospho-glucuronosyl-transferase. The in vivo study was performed by analyzing urines collected after the oral administration of methandienone with and without the co-administration of ketoconazole. Methandienone and its metabolites were determined by liquid chromatography-mass spectrometry-based techniques after sample pre-treatment including an enzymatic hydrolysis step (performed only for the investigation on phase II metabolism) and liquid/liquid extraction with t-butyl methyl-ether. The results from the in vitro experiments showed that the formation of the hydroxylated and dehydrogenated metabolites was significantly reduced in the presence of itraconazole, ketoconazole, miconazole and nefazodone, whereas the production of the 18-nor-hydroxylated metabolites and glucuronidation reactions was reduced significantly only in the presence of ketoconazole and miconazole. The analysis of the post-administration samples confirmed the in vitro observations, validating the hypothesis that drug-drug interaction may cause significant alterations in the metabolic profile of banned drugs, making their detection during doping control tests more challenging. This article is protected by copyright. All rights reserved.

Folate-containing reduction-sensitive lipid-polymer hybrid nanoparticles for targeted delivery of doxorubicin.

PubMed

Wu, Bo; Yu, Ping; Cui, Can; Wu, Ming; Zhang, Yang; Liu, Lei; Wang, Cai-Xia; Zhuo, Ren-Xi; Huang, Shi-Wen

2015-04-01

The development and evaluation of folate-targeted and reduction-triggered biodegradable nanoparticles are introduced to the research on targeted delivery of doxorubicin (DOX). This type of folate-targeted lipid-polymer hybrid nanoparticles (FLPNPs) is comprised of a poly(D,L-lactide-co-glycolide) (PLGA) core, a soybean lecithin monolayer, a monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16) reduction-sensitive shell, and a folic acid-targeted ligand. FLPNPs exhibited high size stability but fast disassembly in a simulated cancer cell reductive environment. The experiments on the release process in vitro revealed that as a reduction-sensitive drug delivery system, FLPNPs released DOX faster in the presence of 10 mM dithiothreitol (DTT). Results from flow cytometry, confocal image and in vitro cytotoxicity assays revealed that FLPNPs further enhanced cell uptake and generated higher cytotoxicity against human epidermoid carcinoma in the oral cavity than non-targeted redox-sensitive and targeted redox-insensitive controls. Furthermore, in vivo animal experiments demonstrated that systemic administration of DOX-loaded FLPNPs remarkably reduced tumor growth. Experiments on biodistribution of DOX-loaded FLPNPs showed that an increasing amount of DOX accumulated in the tumor. Therefore, FLPNPs formulations have proved to be a stable, controllable and targeted anticancer drug delivery system.

An in vitro and in vivo evaluation of new potential trans-sialidase inhibitors of Trypanosoma cruzi predicted by a computational drug repositioning method.

PubMed

Lara-Ramirez, Edgar E; López-Cedillo, Julio Cesar; Nogueda-Torres, Benjamin; Kashif, Muhammad; Garcia-Perez, Carlos; Bocanegra-Garcia, Virgilio; Agusti, Rosalía; Uhrig, María Laura; Rivera, Gildardo

2017-05-26

Chagas disease is one of the most important neglected parasitic diseases afflicting developed and undeveloped countries. There are currently limited options for inexpensive and secure pharmacological treatment. In this study, we employed a structure-based virtual screening protocol for 3180 FDA-approved drugs for repositioning of them as potential trans-sialidase inhibitors. In vitro and in vivo evaluations were performed for the selected drugs against trypomastigotes from the INC-5 and NINOA strains of T. cruzi. Also, inhibition of sialylation by the trans-sialidase enzyme reaction was evaluated using high-performance anion-exchange chromatography with pulse amperometric detection to confirm the mechanism of action. Results from the computational study showed 38 top drugs with the best binding-energies. Four compounds with antihistaminic, anti-hypertensive, and antibiotic properties showed better trypanocidal effects (LC 50 range = 4.5-25.8 μg/mL) than the reference drugs, nifurtimox and benznidazole (LC 50 range = 36.1-46.8 μg/mL) in both strains in the in vitro model. The anti-inflammatory, sulfasalazine showed moderate inhibition (37.6%) of sialylation in a trans-sialidase enzyme inhibition reaction. Sulfasalazine also showed the best trypanocidal effects in short-term in vivo experiments on infected mice. This study suggests for the first time that the anti-inflammatory sulfasalazine could be used as a lead compound to develop new trans-sialidase inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Using acoustic cavitation to enhance chemotherapy of DOX liposomes: experiment in vitro and in vivo.

PubMed

Zhao, Ying-Zheng; Dai, Dan-Dan; Lu, Cui-Tao; Lv, Hai-Feng; Zhang, Yan; Li, Xing; Li, Wen-Feng; Wu, Yan; Jiang, Lei; Li, Xiao-Kun; Huang, Pin-Tong; Chen, Li-Juan; Lin, Min

2012-09-01

Experiments in vitro and in vivo were designed to investigate tumor growth inhibition of chemotherapeutics-loaded liposomes enhanced by acoustic cavitation. Doxorubicin-loaded liposomes (DOX liposomes) were used in experiments to investigate acoustic cavitation mediated effects on cell viability and chemotherapeutic function. The influence of lingering sensitive period after acoustic cavitation on tumor inhibition was also investigated. Animal experiment was carried out to verify the practicability of this technique in vivo. From experiment results, blank phospholipid-based microbubbles (PBM) combined with ultrasound (US) at intensity below 0.3 W/cm² could produce acoustic cavitation which maintained cell viability at high level. Compared with DOX solution, DOX liposomes combined with acoustic cavitation exerted effective tumor inhibition in vitro and in vivo. The lingering sensitive period after acoustic cavitation could also enhance the susceptibility of tumor to chemotherapeutic drugs. DOX liposomes could also exert certain tumor inhibition under preliminary acoustic cavitation. Acoustic cavitation could enhance the absorption efficiency of DOX liposomes, which could be used to reduce DOX adverse effect on normal organs in clinical chemotherapy.

A new low-profile visualized intraluminal support (LVIS) device, LVIS Blue: laboratory comparison between old and new LVIS.

PubMed

Chung, Joonho; Matsuda, Yoshikazu; Nelson, Jessica; Keigher, Kiffon; Lopes, Demetrius K

2018-01-01

Objectives The Low-profile Visualized Intraluminal Support (LVIS) device is a flexible intracranial stent. The first generation of this system had significant challenges in consistently providing good wall apposition and aneurysm neck coverage. A new modified LVIS, LVIS Blue (Blue), has been developed to address these issues. The purpose of this study is to report a laboratory comparison of wall apposition and aneurysm neck coverage between the original LVIS and Blue. Methods In bench-top experiments, we noted the visual appearance of the devices and evaluated changes in stent cell angles, neck coverage surface area (%), and stent cell crossing profile for microcatheters using a fusiform aneurysm model. Our in vitro experiments included evaluation of wall apposition and aneurysm neck coverage of the devices under direct radiographic visualization. Results Blue showed three definite different zones (a mid-zone, a high-density zone, and a transitional zone) and higher metal coverage in the straight fusiform aneurysm model compared to LVIS. Two commercially available microcatheters easily crossed the stent cell at the greater curvature for both devices. In in vitro experiments, Blue showed better wall apposition in tortuous arteries and achieved higher neck coverage in the bifurcation aneurysm compared to LVIS. Discussion Blue achieved better wall apposition in tortuous arteries and higher aneurysm neck coverage (higher metal-to-artery ratio) in bifurcation aneurysms than LVIS. Our results may provide informative physical properties of LVIS and Blue to be expected when those are used for stent-assisted coil embolization of a large-giant fusiform aneurysm or a bifurcation saccular aneurysm clinically.

Effect of high oxygen modified atmosphere packaging on microbial growth and sensorial qualities of fresh-cut produce.

PubMed

Jacxsens, L; Devlieghere, F; Van der Steen, C; Debevere, J

2001-12-30

The application of High Oxygen Atmospheres (HOA) (i.e. > 70% O2) for packaging ready-to-eat vegetables was evaluated as an alternative technique for low O2 Equilibrium Modified Atmosphere (EMA) packaging (3% O2-5% CO2-balance N2) for respiring products. Comparative experiments between both techniques were performed in-vitro and in-vivo. Typical spoilage causing microorganisms (Pseudomonas fluorescens, Candida lambica), the moulds Botrytis cinerea, Aspergillus flavus and the opportunistic psychrotrophic human pathogenic microorganism associated with refrigerated minimally processed vegetables. Aeromonas caviae (HG4), showed a retarded growth during the conducted in-vitro studies at 4 degrees C in 70%, 80% and 95% O2 as examples of HOA compared to the in-vitro experiments in 5% O2 (as example of EMA packaging) and the effect was more pronounced in 95% O2. The effect of the high O2-concentrations on the human pathogen Listeria monocytogenes resulted in an extended lag phase (95% O2). The plant pathogen Erwinia carotovora was increasingly stimulated by increasing high O2-concentrations. During a storage experiment of three types of ready-to-eat vegetables (mushroom slices, grated celeriac and shredded chicory endive), which are sensitive to enzymatic browning and microbial spoilage, the effect of EMA and HOA (95% O2-5% N2) on their quality and shelf life was compared. High O2 atmospheres were found to be particularly effective in inhibiting enzymatic browning of the tested vegetables. Also, the microbial quality was better as a reduction in yeast growth was observed. The HOA can be applied as an alternative for low O2 modified atmospheres for some specific types of ready-to-eat vegetables, sensitive to enzymatic browning and spoilage by yeasts.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

PubMed

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Reliability of In Vitro Methods used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial.

PubMed

Nichols, John; Fay, Kellie; Bernhard, Mary Jo; Bischof, Ina; Davis, John; Halder, Marlies; Hu, Jing; Johanning, Karla; Laue, Heike; Nabb, Diane; Schlechtriem, Christian; Segner, Helmut; Swintek, Joe; Weeks, John; Embry, Michelle

2018-05-14

In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intra-laboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, while inter-laboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO,INT; L/d/kg fish), both assays yielded similar levels of activity (< 4-fold difference for all chemicals). Hepatic clearance rates (CLH; L/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay.

PubMed

Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects. Copyright © 2010 Elsevier Inc. All rights reserved.

Novel gadopentetic acid-doped silica nanoparticles conjugated with YPSMA-1 targeting prostate cancer for MR imaging: an in vitro study.

PubMed

Jiang, Wei; He, Xiaojing; Fang, Huiying; Zhou, Xue; Ran, Haitao; Guo, Dajing

2018-05-05

The early diagnosis of prostate cancer (PCa) is particularly important for reducing its high mortality rate. With the development of molecular magnetic resonance imaging (MRI), early diagnosis via non-invasive imaging has become possible. In this study, gadopentetic acid (GA)-doped silica (Gd@SiO 2 ) was first synthesized by a reverse microemulsion method, and amino and carboxyl groups were then successively introduced onto the surface of this Gd@SiO 2 . After these steps, a monoclonal antibody (YPSMA-1) to prostate-specific membrane antigen (PSMA) was conjugated with carboxyl-modified Gd@SiO 2 (Gd@SiO 2 -COOH) nanoparticles (NPs) by the carbodiimide method. Gd@SiO 2 -Ab NPs were thus obtained as specific MR contrast agents for PCa-targeted imaging. Transmission electron microscopy showed that the Gd@SiO 2 -Ab NPs exhibited a dispersed spherical morphology with a relatively uniform size distribution. The Gd@SiO 2 -Ab NPs showed high stability and high the longitudinal relaxation rate (r 1 ). Cell-targeting experiments in vitro demonstrated the high potential of the synthesized NPs to target PSMA receptor-positive PCa cells. In vitro cytotoxicity assays showed that the Gd@SiO 2 -Ab NPs exhibited good biological safety. These results suggest that the synthesized Gd@SiO 2 -Ab NPs have great potential as specific MR contrast agents for PSMA receptor-positive PCa cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls.

PubMed

Alomar, M; Tasiaux, H; Remacle, S; George, F; Paul, D; Donnay, I

2008-08-01

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex ratio of the embryos. Moreover, those parameters might be related.

Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection.

PubMed

Martínez, Fernando; Daròs, José-Antonio

2014-09-01

The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells. In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Fibroblast-mediated in vivo and in vitro growth promotion of tumorigenic rat thyroid carcinoma cells but not normal Fisher rat thyroid follicular cells.

PubMed

Saitoh, Ohki; Mitsutake, Norisato; Nakayama, Toshiyuki; Nagayama, Yuji

2009-07-01

It is known that genetic abnormalities in oncogenes and/or tumor suppressor genes promote carcinogenesis. Numerous recent articles, however, have demonstrated that epithelial-stromal interaction also plays a critical role for initiation and progression of carcinoma cells. Furthermore, ionizing radiation induces alterations in the tissue microenvironments that promote carcinogenesis. There is little or no information on epithelial-stromal interaction in thyroid carcinoma cells. The objective of this study was to determine if epithelial-stromal interaction influenced the growth of thyroid carcinoma cells in vivo and in vitro and to determine if radiation had added or interacting effects. Normal Fisher rat thyroid follicular cells (FRTL5 cells) and tumorigenic rat thyroid carcinoma cells (FRTL-Tc cells) derived from FRTL5 cells were employed. The cells were injected into thyroids or subcutaneously into left flanks of rats alone or in combination with skin-derived fibroblasts. In groups of rats, fibroblasts were irradiated with 0.1 or 4 Gy x-ray 3 days before inoculation. In vitro growth of FRTL-Tc and FRTL-5 cells were evaluated using the fibroblast-conditioned medium and in a co-culture system with fibroblasts. The in vivo experiments demonstrated that FRTL-Tc cells injected intrathyroidally grew faster than those injected subcutaneously, and that admixed fibroblasts enhanced growth of subcutaneous FRTL-Tc tumors, indicating that the intrathyroidal milieu, particularly in the presence of fibroblasts, confer growth-promoting advantage to thyroid carcinoma cells. This in vivo growth-promoting effect of fibroblasts on FRTL-Tc cells was duplicated in the in vitro experiments using the fibroblast-conditioned medium. Thus, our data demonstrate that this effect is mediated by soluble factor(s), is reversible, and is comparable to that of 10% fetal bovine serum. However, normal FRTL5 cells did not respond to the fibroblast-conditioned medium. Furthermore, high- and low-dose irradiation enhanced and suppressed, respectively, the in vivo fibroblast-mediated growth promotion. This effect was, however, not observed in the in vitro experiment with conditioned medium or even that allowing cell-cell contact. The intrathyroidal stromal microenvironments, particularly fibroblasts, appear to enhance the growth of thyroid carcinomas through soluble factor(s), which is modulated differently by high- and low-dose irradiation. To our knowledge this is the first study to show epithelial-stromal interaction in thyroid carcinoma.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

PubMed

Paes, V M; Vieira, L A; Correia, H H V; Sa, N A R; Moura, A A A; Sales, A D; Rodrigues, A P R; Magalhães-Padilha, D M; Santos, F W; Apgar, G A; Campello, C C; Camargo, L S A; Figueiredo, J R

2016-09-01

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC. Copyright © 2016 Elsevier Inc. All rights reserved.

Engineered Resistant-Starch (ERS) Diet Shapes Colon Microbiota Profile in Parallel with the Retardation of Tumor Growth in In Vitro and In Vivo Pancreatic Cancer Models.

PubMed

Panebianco, Concetta; Adamberg, Kaarel; Adamberg, Signe; Saracino, Chiara; Jaagura, Madis; Kolk, Kaia; Di Chio, Anna Grazia; Graziano, Paolo; Vilu, Raivo; Pazienza, Valerio

2017-03-27

Pancreatic cancer (PC) is ranked as the fourth leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, a modest impact on the outcome of the disease is observed so far. We have previously demonstrated that short-term fasting cycles have the potential to improve the efficacy of chemotherapy against PC. The aim of this study was to assess the effect of an engineered resistant-starch (ERS) mimicking diet on the growth of cancer cell lines in vitro, on the composition of fecal microbiota, and on tumor growth in an in vivo pancreatic cancer mouse xenograft model. BxPC-3, MIA PaCa-2 and PANC-1 cells were cultured in the control, and in the ERS-mimicking diet culturing condition, to evaluate tumor growth and proliferation pathways. Pancreatic cancer xenograft mice were subjected to an ERS diet to assess tumor volume and weight as compared to mice fed with a control diet. The composition and activity of fecal microbiota were further analyzed in growth experiments by isothermal microcalorimetry. Pancreatic cancer cells cultured in an ERS diet-mimicking medium showed decreased levels of phospho-ERK1/2 (extracellular signal-regulated kinase proteins) and phospho-mTOR (mammalian target of rapamycin) levels, as compared to those cultured in standard medium. Consistently, xenograft pancreatic cancer mice subjected to an ERS diet displayed significant retardation in tumor growth. In in vitro growth experiments, the fecal microbial cultures from mice fed with an ERS diet showed enhanced growth on residual substrates, higher production of formate and lactate, and decreased amounts of propionate, compared to fecal microbiota from mice fed with the control diet. A positive effect of the ERS diet on composition and metabolism of mouse fecal microbiota shown in vitro is associated with the decrease of tumor progression in the in vivo PC xenograft mouse model. These results suggest that engineered dietary interventions could be supportive as a synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients.

Engineered Resistant-Starch (ERS) Diet Shapes Colon Microbiota Profile in Parallel with the Retardation of Tumor Growth in In Vitro and In Vivo Pancreatic Cancer Models

PubMed Central

Panebianco, Concetta; Adamberg, Kaarel; Adamberg, Signe; Saracino, Chiara; Jaagura, Madis; Kolk, Kaia; Di Chio, Anna Grazia; Graziano, Paolo; Vilu, Raivo; Pazienza, Valerio

2017-01-01

Background/aims: Pancreatic cancer (PC) is ranked as the fourth leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, a modest impact on the outcome of the disease is observed so far. We have previously demonstrated that short-term fasting cycles have the potential to improve the efficacy of chemotherapy against PC. The aim of this study was to assess the effect of an engineered resistant-starch (ERS) mimicking diet on the growth of cancer cell lines in vitro, on the composition of fecal microbiota, and on tumor growth in an in vivo pancreatic cancer mouse xenograft model. Materials and Methods: BxPC-3, MIA PaCa-2 and PANC-1 cells were cultured in the control, and in the ERS-mimicking diet culturing condition, to evaluate tumor growth and proliferation pathways. Pancreatic cancer xenograft mice were subjected to an ERS diet to assess tumor volume and weight as compared to mice fed with a control diet. The composition and activity of fecal microbiota were further analyzed in growth experiments by isothermal microcalorimetry. Results: Pancreatic cancer cells cultured in an ERS diet-mimicking medium showed decreased levels of phospho-ERK1/2 (extracellular signal-regulated kinase proteins) and phospho-mTOR (mammalian target of rapamycin) levels, as compared to those cultured in standard medium. Consistently, xenograft pancreatic cancer mice subjected to an ERS diet displayed significant retardation in tumor growth. In in vitro growth experiments, the fecal microbial cultures from mice fed with an ERS diet showed enhanced growth on residual substrates, higher production of formate and lactate, and decreased amounts of propionate, compared to fecal microbiota from mice fed with the control diet. Conclusion: A positive effect of the ERS diet on composition and metabolism of mouse fecal microbiota shown in vitro is associated with the decrease of tumor progression in the in vivo PC xenograft mouse model. These results suggest that engineered dietary interventions could be supportive as a synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients. PMID:28346394

The effect of in vivo and in vitro irradiation (25 Gy) on the subsequent in vitro growth of satellite cells

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Schultz, E.; Cassens, R. G.

1996-01-01

The effect of in vivo and in vitro irradiation on subsequent satellite cell growth, in vitro, was investigated to ascertain the ability of a 25 Gy dose to inhibit satellite cell proliferation. Satellite cells were isolated from the left (irradiated) and right (non-irradiated) Pectoralis thoracicus of two-week-old tom turkeys 16 h (n=3) and seven weeks (n=2) after the left Pectoralis thoracicus had been irradiated (25 Gy). Satellite cells isolated from the irradiated and non-irradiated muscles exhibited similar (P>0.10) in vitro proliferation indicating that a population of satellite cells survived an in vivo dose of 25 Gy. In additional experiments, satellite cell cultures derived from tom turkey Pectoralis thoracicus were irradiated (25 Gy) in vitro. The number of satellite cells did not (P>0.05) increase in irradiated cultures for 134 h following irradiation, while satellite cells in non-irradiated cultures proliferated (P<0.05) over this time. At later time periods, satellite cell number increased (P<0.05) in irradiated cultures indicating that a population of satellite cells survived irradiation. The results of these in vitro experiments suggest that a 25 Gy dose of irradiation does not abolish satellite cell divisions in the turkey Pectoralis thoracicus.

The anxiety of Taiwanese women with or without continuity treatment after previous in vitro fertilisation failure.

PubMed

Su, Tsann-Juu; Tzeng, Ya-Ling; Kuo, Pi-Chao

2011-08-01

To compare the anxiety levels of Taiwanese women who continued with in vitro fertilisation treatment and those who discontinued treatment post-in vitro fertilisation failure. In vitro fertilisation is perceived as the last resort of infertility treatment. The impact of unsuccessful in vitro fertilisation treatment on psychological function has been documented; however, research comparing the levels of anxiety of women who cease and those who continue in vitro fertilisation post-failure is scant. A cross-sectional comparative study design was used. Fifty-eight women in whom in vitro fertilisation had failed within the previous year were recruited to this study from a medical centre in northern Taiwan; 34 women continued treatment and 24 discontinued treatment. The State-Trait Anxiety Inventory was used to assess their levels of anxiety. Women in the group who continued treatment exhibited higher state and trait anxiety (TA) than women in the group who discontinued treatment (p < 0·005). The number and frequency of in vitro fertilisation cycles were significantly higher in the group who continued treatment than in those who did not. A strong positive correlation between state and TA (r = 0·8, p < 0·01) existed in both groups. Both groups exhibited considerable levels of anxiety; however, the women who continued in vitro fertilisation treatment had higher levels of anxiety than those who discontinued treatment. The level of anxiety of women who decide to continue in vitro fertilisation treatment should be assessed as early as possible and counselling services provided to women who experience in vitro fertilisation failure should concentrate more on relieving psychological distress. One year after discontinuing treatment, some women still experience considerable anxiety; therefore, the care and assistance provided to these women need to be continually evaluated. © 2011 Blackwell Publishing Ltd.

Anthelmintic activity of Cocos nucifera L. against sheep gastrointestinal nematodes.

PubMed

Oliveira, L M B; Bevilaqua, C M L; Costa, C T C; Macedo, I T F; Barros, R S; Rodrigues, A C M; Camurça-Vasconcelos, A L F; Morais, S M; Lima, Y C; Vieira, L S; Navarro, A M C

2009-01-22

The development of anthelmintic resistance has made the search for alternatives to control gastrointestinal nematodes of small ruminants imperative. Among these alternatives are several medicinal plants traditionally used as anthelmintics. This work evaluated the efficacy of Cocos nucifera fruit on sheep gastrointestinal parasites. The ethyl acetate extract obtained from the liquid of green coconut husk fiber (LGCHF) was submitted to in vitro and in vivo tests. The in vitro assay was based on egg hatching (EHT) and larval development tests (LDT) with Haemonchus contortus. The concentrations tested in the EHT were 0.31, 0.62, 1.25, 2.5 and 5 mg ml(-1), while in the LDT they were 5, 10, 20, 40 and 80 mg ml(-1). The in vivo assay was a controlled test. In this experiment, 18 sheep infected with gastrointestinal nematodes were divided into three groups (n=6), with the following doses administered: G1-400 mg kg(-1) LGCHF ethyl acetate extract, G2-0.2 mg kg(-1) moxidectin (Cydectin) and G3-3% DMSO. The worm burden was analyzed. The results of the in vitro and in vivo tests were submitted to ANOVA and analyzed by the Tukey and Kruskal-Wallis tests, respectively. The extract efficacy in the EHT and LDT, at the highest concentrations tested, was 100% on egg hatching and 99.77% on larval development. The parameters evaluated in the controlled test were not statistically different, showing that despite the significant results of the in vitro tests, the LGCHF ethyl acetate extract showed no activity against sheep gastrointestinal nematodes.

Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity.

PubMed

Elferink, M G L; Olinga, P; Draaisma, A L; Merema, M T; Bauerschmidt, S; Polman, J; Schoonen, W G; Groothuis, G M M

2008-06-15

The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such as Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl(4), fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.

Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elferink, M.G.L.; Olinga, P.; Draaisma, A.L.

2008-06-15

The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such asmore » Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl{sub 4}, fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.« less

Toxicity of polymeric nanoparticles in vivo and in vitro

NASA Astrophysics Data System (ADS)

Voigt, Nadine; Henrich-Noack, Petra; Kockentiedt, Sarah; Hintz, Werner; Tomas, Jürgen; Sabel, Bernhard A.

2014-06-01

Polybutylcyanoacrylate nanoparticles (PBCA NPs) are candidates for a drug delivery system, which can cross the blood-brain barrier (BBB). Because little is known about their toxicity, we exposed cells to PBCA NPs in vitro and in vivo and monitored their life and death assays. PBCA NPs were fabricated with different surfactants according to the mini-emulsion technique. Viabilities of HeLa and HEK293 cells after NP incubation were quantified by analysing cellular metabolic activity (MTT-test). We then repetitively injected i.v. rhodamine-labelled PBCA NP variations into rats and monitored the survival and morphology of retrogradely labelled neurons by in vivo confocal neuroimaging (ICON) for five weeks. To test for carrier-efficacy and safety, PBCA NPs loaded with Kyotorphin were injected in rats, and a hot plate test was used to quantify analgesic effects. In vitro, we found dose-dependent cell death which was, however, only detectable at very high doses and mainly seen in the cultures incubated with NPs fabricated with the tensids SDS and Tween. However, the in vivo experiments did not show any NP-induced neuronal death, even with particles which were toxic at high dose in vitro, i.e. NPs with Tween and SDS. The increased pain threshold at the hot plate test demonstrated that PBCA NPs are able to cross the BBB and thus comprise a useful tool for drug delivery into the central nervous system (CNS). Our findings showing that different nanoparticle formulations are non-toxic have important implications for the value of NP engineering approaches in medicine.

Geissoschizine methyl ether N-oxide, a new alkaloid with antiacetylcholinesterase activity from Uncaria rhynchophylla.

PubMed

Jiang, Wei-Wei; Su, Jia; Wu, Xing-De; He, Juan; Peng, Li-Yan; Cheng, Xiao; Zhao, Qin-Shi

2015-01-01

Geissoschizine methyl ether N-oxide, a new oxindole alkaloid, along with 14 known alkaloids, was isolated from the aerial part of Uncaria rhynchophylla. Their structures were identified by comprehensive spectral methods, including 2D NMR experiments, and confirmed by comparing with the literature data. In vitro acetylcholinesterase (AChE) inhibitory activity assay showed that the new compound exhibited anti-AChE activity with IC₅₀ value of 23.4 μM.

Cor a 14, the allergenic 2S albumin from hazelnut, is highly thermostable and resistant to gastrointestinal digestion

PubMed Central

Pfeifer, Sabine; Bublin, Merima; Dubiela, Pawel; Hummel, Karin; Wortmann, Judith; Hofer, Gerhard; Keller, Walter; Radauer, Christian

2015-01-01

Scope Allergens from nuts frequently induce severe allergic reactions in sensitive individuals. The aim of this study was to elucidate the physicochemical characteristics of natural Cor a 14, the 2S albumin from hazelnut. Methods and results Cor a 14 was purified from raw hazelnuts using a combination of precipitation and chromatographic techniques. The protein was analyzed using gel electrophoresis, MS, and far‐UV circular dichroism (CD) analyses. The immunoglobulin E (IgE) binding of native, heat‐treated, and in vitro digested Cor a 14 was studied. We identified two different Cor a 14 isoforms and showed microclipping at the C‐terminus. CD spectra at room temperature showed the typical characteristics of 2S albumins, and temperatures of more than 80°C were required to start unfolding of Cor a 14 demonstrating its high stability to heat treatment. In vitro digestion experiments revealed that Cor a 14 is resistant to proteolytic degradation. Native and heat‐treated protein was recognized by sera from hazelnut allergic patients. However, denaturation of the allergen led to significantly reduced IgE binding. Conclusion We identified two different isoforms of Cor a 14 displaying high stability under heating and gastric and duodenal conditions. Data from IgE‐binding experiments revealed the existence of both, linear and conformational epitopes. PMID:26178695

Chemistry-driven structural alterations in short-term retrieved ceramic-on-metal hip implants: Evidence for in vivo incompatibility between ceramic and metal counterparts.

PubMed

Zhu, Wenliang; Pezzotti, Giuseppe; Boffelli, Marco; Chotanaphuti, Thanainit; Khuangsirikul, Saradej; Sugano, Nobuhiko

2017-08-01

Ceramic-on-metal (CoM) hip implants were reported to experience lower wear rates in vitro as compared to metal-on-metal (MoM) bearings, thus hinting metal-ion release at lower levels in vivo. In this article, we show a spectroscopic study of two short-term retrieval cases of zirconia-toughened alumina (ZTA) femoral heads belonging to CoM hip prostheses, which instead showed poor wear performances in vivo. Metal contamination and abnormally high fractions of tetragonal-to-monoclinic (t→m) polymorphic transformation of the zirconia phase could be found on both ZTA heads, which contrasted with the optimistic predictions of in vitro experiments. At the molecular scale, incorporation of metal ions into the ceramic lattices could be recognized as due to frictionally assisted phenomena occurring at the ceramic surface. Driven by abnormal friction, diffusion of metal ions induced lattice shrinkage in the zirconia phases, while residual stress fields became stored at the surface of the femoral head. Diffusional alterations destabilized the chemistry of the ceramic surface and resulted in an abnormal increase in t→m phase transformation in vivo. Frictionally driven metal transfer to the ceramic lattice thus hinders the in vivo performance of CoM prostheses. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1469-1480, 2017. © 2016 Wiley Periodicals, Inc.

In vitro biocompatibility of EPM and EPDM rubbers.

PubMed

Mast, F; Hoschtitzky, J A; Van Blitterswijk, C A; Huysmans, H A

1997-01-01

The in vitro toxicity of two EPDM rubbers (K 778 and K 4802) and one EPM rubber (K 740) was tested using human fibroblasts. The modulus of elasticity of each rubber was varied by exposure to different amounts of electron-beam radiation (0, 5 and 10 Mrad). The short-term in vitro toxicity was tested by culturing cells on polymer films. The long-term effect of ageing was simulated by growing fibroblasts in nutrient media prepared from extracts of heat-exposed materials. Cell cultures were studied both quantitatively and (ultra) structurally. Growth curves obtained in the toxicity test did not differ significantly from control values at any day of observation, and also showed that electron-beam radiation did not alter the biocompatibility. The same results were found for all but one material in the artificial ageing test. The number of cells in the K4802/10 Mrad extraction medium was decreased. Ultrastructurally no gross deviations from normal morphology were observed, either in the direct contact test or in the artificial ageing test. The most characteristic feature was a somewhat dilated endoplasmic reticulum. In summary, the in vitro biocompatibility of EPDM-rubbers as observed in this study is satisfactory and motivates further investigation of their biocompatibility in animal experiments.

A preliminary evaluation of self-made nanobubble in contrast-enhanced ultrasound imaging

NASA Astrophysics Data System (ADS)

Li, Chunfang; Wu, Kaizhi; Li, Jing; Liu, Haijuan; Zhou, Qibing; Ding, Mingyue

2014-03-01

Nanoscale bubbles (nanobubbles) have been reported to improve contrast in tumor-targeted ultrasound imaging due to the enhanced permeation and retention effects at tumor vascular leaks. In this work, a self-made nanobubble ultrasound contrast agent was preliminarily characterized and evaluated in-vitro and in-vivo. Fundamental properties such as morphology appearance, size distribution, zeta potential, bubble concentration (bubble numbers per milliliter contrast agent suspension) and the stability of nanobubbles were assessed by light microscope and particle sizing analysis. Then the concentration intensity curve and time intensity curves (TICs) were acquired by ultrasound imaging experiment in-vitro. Finally, the contrast-enhanced ultrasonography was performed on rat to investigate the procedure of liver perfusion. The results showed that the nanobubbles had good shape and uniform distribution with the average diameter of 507.9 nm, polydispersity index (PDI) of 0.527, and zeta potential of -19.17 mV. Significant contrast enhancement was observed in in-vitro ultrasound imaging, demonstrating that the self-made nanobubbles can enhance the contrast effect of ultrasound imaging efficiently in-vitro. Slightly contrast enhancement was observed in in-vivo ultrasound imaging, indicating that the nanobubbles are not stable enough in-vivo. Future work will be focused on improving the ultrasonic imaging performance, stability, and antibody binding of the nanoscale ultrasound contrast agent.

Development of performance matrix for generic product equivalence of acyclovir topical creams.

PubMed

Krishnaiah, Yellela S R; Xu, Xiaoming; Rahman, Ziyaur; Yang, Yang; Katragadda, Usha; Lionberger, Robert; Peters, John R; Uhl, Kathleen; Khan, Mansoor A

2014-11-20

The effect of process variability on physicochemical characteristics and in vitro performance of qualitatively (Q1) and quantitatively (Q2) equivalent generic acyclovir topical dermatological creams was investigated to develop a matrix of standards for determining their in vitro bioequivalence with reference listed drug (RLD) product (Zovirax®). A fractional factorial design of experiment (DOE) with triplicate center point was used to create 11 acyclovir cream formulations with manufacturing variables such as pH of aqueous phase, emulsification time, homogenization speed, and emulsification temperature. Three more formulations (F-12-F-14) with drug particle size representing RLD were also prepared where the pH of the final product was adjusted. The formulations were subjected to physicochemical characterization (drug particle size, spreadability, viscosity, pH, and drug concentration in aqueous phase) and in vitro drug release studies against RLD. The results demonstrated that DOE formulations were structurally and functionally (e.g., drug release) similar (Q3) to RLD. Moreover, in vitro drug permeation studies showed that extent of drug bioavailability/retention in human epidermis from F-12-F-14 were similar to RLD, although differed in rate of permeation. The results suggested generic acyclovir creams can be manufactured to obtain identical performance as that of RLD with Q1/Q2/Q3. Published by Elsevier B.V.

Modeling the Effect of Temperature and Potential on the In Vitro Corrosion Performance of Biomedical Hydroxyapatite Coatings

NASA Astrophysics Data System (ADS)

Coşkun, M. İbrahim; Karahan, İsmail H.; Yücel, Yasin; Golden, Teresa D.

2016-10-01

CoCrMo biomedical alloys were coated with a hydroxyapatite layer to improve biocompatibility and in vitro corrosion performance. A fast electrodeposition process was completed in 5 minutes for the hydroxyapatite coating. Effect of the solution temperature and applied potential on the in vitro corrosion performance of the hydroxyapatite coatings was modeled by response surface methodology (RSM) coupled with central composite design (CCD). A 5-level-2-factor experimental plan designed by CCD was used; the experimental plan contained 13 coating experiments with a temperature range from 283 K to 347 K (10 °C to 74 °C) and potential range from -1.2 to -1.9 V. Corrosion potential ( E corr) of the coatings in a simulated body fluid solution was chosen as response for the model. Predicted and experimental values fitted well with an R 2 value of 0.9481. Response surface plots of the impedance and polarization resistance ( R P) were investigated. Optimized parameters for electrodeposition of hydroxyapatite were determined by RSM as solution temperature of 305.48 K (32.33 °C) and potential of -1.55 V. Hydroxyapatite coatings fabricated at optimized parameters showed excellent crystal formation and high in vitro corrosion resistance.

Fertilisation in the horse and paracrine signalling in the oviduct.

PubMed

Goudet, Ghylène

2011-01-01

The mammalian oviduct plays a crucial role in the preparation of gametes for fertilisation (transport and final maturation) and fertilisation itself. An increasing number of studies offers a comprehensive overview of the functions of the oviduct and its secretions, but this topic has had limited investigation in the horse. Limited data are available on the final oocyte maturation in the equine oviduct. However, in vitro and in vivo systems have been established to analyse the influence of equine oviduct epithelial cells (OEC) during maturation on the potential of oocytes for fertilisation and development. Most studies focus on the role of the oviduct in equine sperm function, such as spermatozoa transport, attachment to oviduct epithelium, viability, motility and capacitation. Moreover, some possible candidate molecules for sperm-oviducal interactions have been identified in the horse. Finally, the low efficiency of conventional in vitro fertilisation and the in vivo fertilisation of equine oocytes transferred into the oviduct of an inseminated mare predicted an influence of oviduct in equine fertilisation. Actually, in vivo and in vitro experiments demonstrated a role of the oviduct in equine fertilisation. Moreover, recent studies showed a beneficial effect of homologous and heterologous OEC on equine in vitro fertilisation, and some candidate molecules have been studied.

In vitro neutralization of HCV by goat antibodies against peptides encompassing regions downstream of HVR-1 of E2 glycoprotein.

PubMed

Tabll, Ashraf A; Atef, Khaled; Bader El Din, Noha G; El Abd, Yasmine S; Salem, Ahmed; Sayed, Ahmed A; Dawood, Reham M; Omran, Moataza H; El-Awady, Mostafa K

2014-01-01

This article aims at testing several in vitro systems with various viral sources and cell lines for propagation of HCV to evaluate goat antibodies raised against three E2 epitopes in viral neutralization experiments. Four human cell lines (Huh-7, Huh-7.5, HepG2, and CaCo2) were tested using two different HCV viral sources; Genotype 4 infected sera and J6/JFH HCV cc particles. Neutralization capacity of goat Abs against conserved E2 epitopes; p412 (a.a 412-419), p517 (a.a 517-531), and p430 (a.a 430-447) were examined in the above mentioned in vitro systems. Although infection with patients' sera seems to mimic the in vitro situation, it has limited replication rates as compared with HCV cc particularly in Huh7.5 cells. Non-HCV adapted Huh-7 cells were also found susceptible for transfection with J6/JFH virus but at much slower kinetics. The results of the neutralization assay showed that anti p412 and anti p517 were highly neutralizing to HCVcc. Our data demonstrate that antibodies directed against the viral surface glycoprotein E2 reduced the infectivity of the J6/JFH virus and are promising agents for immunotherapy and HCV vaccine development.

Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

PubMed Central

Ding, Dongqin; Liu, Yongfei; Xu, Yiran; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

2016-01-01

L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product. PMID:27558633

In vitro corrosion resistance of porous NiTi intervertebral fusion devices

NASA Astrophysics Data System (ADS)

Schrooten, Jan; Assad, Michel; Van Humbeeck, Jan; Leroux, Michel A.

2007-02-01

Porous titanium-nickel (PTN) intervertebral fusion devices, produced by self-propagating high-temperature synthesis, represent an alternative to traditional long-term implants in the orthopaedic field. PTN promotes tissue ingrowth and has succeeded short-term and long-term biocompatibility in vivo testing. In this in vitro study, the PTN morphology was characterized using microfocus computer tomography (μCT) in order to calculate the active PTN surface. Potentiodynamic polarization testing was then performed to evaluate the in vitro corrosion resistance of PTN devices in Hanks' based salt solution. Direct coupling experiments of PTN with Ti6Al4V were also performed in order to establish the galvanic corrosion resistance of PTN intervertebral implants in the presence of potential Ti6Al4V supplemental fixation devices. Compared to the behaviour of other orthopaedic biomaterials and solid NiTi devices, PTN devices showed a level of corrosion resistance that is comparable to other NiTi devices and acceptable for the intended orthopaedic application. Further improvement of the corrosion resistance is still possible by specific electrochemical surface treatments.

Biodegradable double-targeted PTX-mPEG-PLGA nanoparticles for ultrasound contrast enhanced imaging and antitumor therapy in vitro.

PubMed

Ma, Jing; Shen, Ming; Xu, Chang Song; Sun, Ying; Duan, You Rong; Du, Lian Fang

2016-11-29

A porous-structure nano-scale ultrasound contrast agent (UCA) was made of monomethoxypoly (ethylene glycol)-poly (lactic-co-glycolic acid) (mPEG-PLGA), and modified by double-targeted antibody: anti-carcinoembryonic antigen (CEA) and anti-carbohydrate antigen 19-9 (CA19-9), as a double-targeted nanoparticles (NPs). Anti-tumor drug paclitaxel (PTX) was encapsulated in the double-targeted nanoparticles (NPs). The morphor and release curve were characterized. We verified a certain anticancer effect of PTX-NPs through cytotoxicity experiments. The cell uptake result showed much more NPs may be facilitated to ingress the cells or tissues with ultrasound (US) or ultrasound targeted microbubble destruction (UTMD) transient sonoporation in vitro. Ultrasound contrast-enhanced images in vitro and in vivo were investigated. Compared with SonoVue, the NPs prolonged imaging time in rabbit kidneys and tumor of nude mice, which make it possible to further enhance anti-tumor effects by extending retention time in the tumor region. The novel double-targeted NPs with the function of ultrasound contrast enhanced imaging and anti-tumor therapy can be a promising way in clinic.

Synthesis of CdTe quantum dot-conjugated CC49 and their application for in vitro imaging of gastric adenocarcinoma cells

NASA Astrophysics Data System (ADS)

Zhang, Yun-Peng; Sun, Peng; Zhang, Xu-Rui; Yang, Wu-Li; Si, Cheng-Shuai

2013-06-01

The purpose of this experiment was to investigate the visible imaging of gastric adenocarcinoma cells in vitro by targeting tumor-associated glycoprotein 72 (TAG-72) with near-infrared quantum dots (QDs). QDs with an emission wavelength of about 550 to 780 nm were conjugated to CC49 monoclonal antibodies against TAG-72, resulting in a probe named as CC49-QDs. A gastric adenocarcinoma cell line (MGC80-3) expressing high levels of TAG-72 was cultured for fluorescence imaging, and a gastric epithelial cell line (GES-1) was used for the negative control group. Transmission electron microscopy indicated that the average diameter of CC49-QDs was 0.2 nm higher compared with that of the primary QDs. Also, fluorescence spectrum analysis indicated that the CC49-QDs did not have different optical properties compared to the primary QDs. Immunohistochemical examination and in vitro fluorescence imaging of the tumors showed that the CC49-QDs probe could bind TAG-72 expressed on MGC80-3 cells.

Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

PubMed

Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

2015-10-01

Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

Application of the moving-actuator type pump as a ventricular assist device: in vitro and in vivo studies.

PubMed

Lee, H S; Rho, Y R; Park, C Y; Hwang, C M; Kim, W G; Sun, K; Choi, M J; Lee, K K; Cheong, J T; Shim, E B; Min, B G

2002-06-01

A moving actuator type pump has been developed as a multifunctional Korean artificial heart (AnyHeart). The pump consists of a moving actuator as an energy converter, right and left sacs, polymer (or mechanical) valves, and a rigid polyurethane housing. The actuator containing a brushless DC motor moves back and forth on an epicyclical gear train to produce a pendular motion, which compresses both sacs alternately. Of its versatile functions of ventricular assist device and total artificial heart use, we have evaluated the system performance as a single or biventricular assist device through in vitro and in vivo experiments. Pump performance and anatomical feasibility were tested using various animals of different sizes. In the case of single ventricular assist device (VAD) use, one of the sacs remained empty and a mini-compliance chamber was attached to either an outflow or inflow port of the unused sac. The in vitro and in vivo studies show acceptable performance and pump behavior. Further extensive study is required to proceed to human application.

Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

PubMed

Guimarães-Camboa, Nuno; Cattaneo, Paola; Sun, Yunfu; Moore-Morris, Thomas; Gu, Yusu; Dalton, Nancy D; Rockenstein, Edward; Masliah, Eliezer; Peterson, Kirk L; Stallcup, William B; Chen, Ju; Evans, Sylvia M

2017-03-02

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of methylmercury on the rat mast cell degranulation

NASA Astrophysics Data System (ADS)

Graevskaya, E. E.; Yasutake, A.; Aramai, R.; Rubin, A. B.

2003-05-01

Methylmercury is the well-known neurotoxicant as weil as a modulator of the immune system. We investigated the effects of MeHg on the rat mast cell degranulation induced by nonimmunological stimuli (the selective liberator of histamine, compound 48/80, and calcium ionophore A23187) both in vivo and in vitro. In 8, 12 and 15 days afterthe final administration of MeHg we observed the suppression of calcium ionophore A23187-and 48/80-induced histamine release, which enhanced with time. In experiments in vitro incubation of peritoneal mast cells with MeHg alone in the dose range 10^{-8} to 10^{-6} did not induce mast cell degranulation, however modified the activation of mast cells by compound 48/80, and calcium ionophore A23187. We observed activation of stimulated secretion by preliminary incubation with low dose of MeHg 10^{-8} M and inhibition by dose of MeHg 10^{-6} M. These results show that MeHg treatment can modify mast cell function in vivo and in vitro and provide insight into the understanding what role this cell has in the pathogenesis of Minamata disease-comlected disorders.

Monitoring protein hydrolysis by pepsin using pH-stat: In vitro gastric digestions in static and dynamic pH conditions.

PubMed

Mat, Damien J L; Cattenoz, Thomas; Souchon, Isabelle; Michon, Camille; Le Feunteun, Steven

2018-01-15

This study intends to demonstrate that acid titration at low pH is very well adapted to the monitoring of pepsin activity. After a description of the underlying principles, this approach was used during in vitro gastric digestions of a model of complex food containing 15wt% of whey proteins, according to both static (2h at pH = 3, Infogest protocol) and dynamic pH conditions (from pH 6.3 down to 2 in 1h). Pepsin activity was quantitatively assessed in all experiments through the calculation of degrees of hydrolysis (DH). Final values of 3.7 and 3.0% were obtained in static and dynamic pH conditions, respectively, and validated using an independent method. Results also show that about 92% of the peptides were detected at pH = 3, and 100% for pH≤2.5. Overall, the proposed approach proved to be very worthy to study protein hydrolysis during in vitro gastric digestions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of biomimetic in vitro fatigue assessment for UHMWPE implant materials.

PubMed

Scholz, Ronja; Knyazeva, Marina; Porchetta, Dario; Wegner, Nils; Senatov, Fedor; Salimon, Alexey; Kaloshkin, Sergey; Walther, Frank

2018-05-26

An important research goal in the field of biomaterials lies in the progressive amendment of in vivo tests with suitable in vitro experiments. Such approaches are gaining more significance nowadays because of an increasing demand on life sciences and the ethical issues bound to the sacrifice of animals for the sake of scientific research. Another advantage of transferring the experiments to the in vitro field is the possibility of accurately control the boundary conditions and experimental parameters in order to reduce the need of validation tests involving animals. With the aim to reduce the amount of needed in vivo studies for this cause, a short-time in vitro test procedure using instrumented load increase tests with superimposed environmental loading has been developed at TUD to assess the mechanical long-term durability of ultra-high molecular weight polyethylene (UHMWPE) under fatigue loading in a biological environment. Copyright © 2018 Elsevier Ltd. All rights reserved.

In vitro investigation of efficacy of new reactivators on OPC inhibited rat brain acetylcholinesterase.

PubMed

Atanasov, Vasil N; Petrova, Iskra; Dishovsky, Christophor

2013-03-25

Organophosphorus compounds (OPC) were developed as warfare nerve agents. They are also widely used as pesticides. The drug therapy of intoxication with OPC includes mainly combination of cholinesterase (ChE) reactivators and cholinolytics. There is no single ChE reactivator having an ability to reactivate sufficiently the inhibited enzyme due to the high variability of chemical structure of the inhibitors. The difficulties in reactivation of ChE activity and slight antidote effect regarding intoxication with some OPC are some of the reasons for continuous efforts to obtain new reactivators of ChE. The aim of the present study was to evaluate the efficacy of some ChE reactivators against OPC intoxication (tabun, paraoxon and dichlorvos) in in vitro experiments and to compare their activity to that known for some currently used oximes (obidoxime, HI-6, 2-PAM). Experiments were carried out using rat brain acetylcholinesterase (AChE). Reactivators showed different activity in the reactivation of rat brain AChE after dichlorvos, paraoxon and tabun inhibition. AChE was easier reactivated after paraoxon treatment. The best effect showed BT-07-4M, obidoxime, TMB-4 and BT-08 from the group of symmetric oximes, and Toxidin, BT-05 and BT-03 from asymmetric compounds. The reactivation of brain AChE inhibited with tabun demonstrated better activity of new compound BT-07-4M, TMB-4 and obidoxime from symmetric oximes, and BT-05 and BT-03 possessing asymmetric structure. All compounds showed low activity toward inhibition of AChE caused by dichlorvos. Comparison of two main structure types (symmetric/asymmetric) showed that the symmetric compounds reactivated better AChE, inhibited with this OPC, than asymmetric ones. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Cytotoxicity of metal and semiconductor nanoparticles indicated by cellular micromotility.

PubMed

Tarantola, Marco; Schneider, David; Sunnick, Eva; Adam, Holger; Pierrat, Sebastien; Rosman, Christina; Breus, Vladimir; Sönnichsen, Carsten; Basché, Thomas; Wegener, Joachim; Janshoff, Andreas

2009-01-27

In the growing field of nanotechnology, there is an urgent need to sensitively determine the toxicity of nanoparticles since many technical and medical applications are based on controlled exposure to particles, that is, as contrast agents or for drug delivery. Before the in vivo implementation, in vitro cell experiments are required to achieve a detailed knowledge of toxicity and biodegradation as a function of the nanoparticles' physical and chemical properties. In this study, we show that the micromotility of animal cells as monitored by electrical cell-substrate impedance analysis (ECIS) is highly suitable to quantify in vitro cytotoxicity of semiconductor quantum dots and gold nanorods. The method is validated by conventional cytotoxicity testing and accompanied by fluorescence and dark-field microscopy to visualize changes in the cytoskeleton integrity and to determine the location of the particles within the cell.

In-vitro ablation of fibrocartilage by XeCl excimer laser

NASA Astrophysics Data System (ADS)

Buchelt, Martin; Papaioannou, Thanassis; Fishbein, Michael C.; Peters, Werner; Beeder, Clain; Grundfest, Warren S.

1991-07-01

A 308 nm excimer laser was employed for ablation of human fibrocartilage. Experiments were conducted in vitro. The tissue response was investigated with respect to dosimetry (ablation rate versus radiant exposure) and thermal effect (thermographic analysis). Irradiation was performed via a 600 um fiber, with radiant exposures ranging between 20mj/mm2 and 80mj/mm2, at 20Hz. The ablation rates were found to range from 3um/pulse to 80um/pulse depending on the radiant exposure and/or the applied pressure on the delivery system. Thermographic analysis, during ablation, revealed maximum average temperatures of about 65 degree(s)C. Similar measurements performed, for the purpose of comparison, with a CW Nd:YAG and a CW CO2 laser showed higher values, of the order of 200 degree(s)C.

Opioids and the immune system: what is their mechanism of action?

PubMed

Eisenstein, Toby K

2011-12-01

There is a significant amount of literature showing that morphine and other opioids modulate immune responses. The findings support many mechanisms by which this may occur. In vitro experiments provide evidence for direct actions of opioids on immune cells using a variety of functional end points. When these drugs are given in vivo, a plethora of immune parameters are also altered. The paper in this issue of the journal by Zhang et al. provides new information on morphine alteration of immune cell subsets in the spleen and thymus of mice and the potential role of glucocorticoids in these observed phenomena. This Commentary reviews the in vitro activities of morphine on leucocytes, as well as other documented mechanisms by which morphine can alter immune function in vivo. © 2011 The Author. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Design, synthesis, molecular modeling and anticholinesterase activity of benzylidene-benzofuran-3-ones containing cyclic amine side chain.

PubMed

Mehrabi, Farzad; Pourshojaei, Yaghoub; Moradi, Alireza; Sharifzadeh, Mohammad; Khosravani, Leila; Sabourian, Reyhaneh; Rahmani-Nezhad, Samira; Mohammadi-Khanaposhtani, Maryam; Mahdavi, Mohammad; Asadipour, Ali; Rahimi, Hamid Reza; Moghimi, Setareh; Foroumadi, Alireza

2017-05-01

A series of 2-benzylidene-benzofuran-3-ones were designed from the structures of Ebselen analogs and aurone derivatives and synthesized in good yields. The target compounds were prepared by the condensation reaction between appropriate benzofuranones with amino alkoxy aldehydes and evaluated as cholinesterase inhibitors by Ellman's method. The in vitro anti-acetylcholinesterase (AChE)/butyrylcholinesterase activities of the synthesized compounds revealed that 7e (IC 50 = 0.045 μM) is the most active compound against AChE. Furthermore, the docking study confirmed the results obtained through in vitro experiments and predicted the possible binding conformation. The anticholinesterase activities of benzylidene-benzofurane-3-ones as aurone analogs revealed that the compounds bearing piperidinylethoxy residue showed better activities against AChE, introducing these compounds for further drug discovery developments. [Formula: see text].

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

PubMed

Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

2016-10-01

Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 10 3 and 10 4 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 10 4 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 10 4 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 10 3 b-ATMSCs/mL (p = 0.051), group 10 4 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 10 4 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

Anti-Toxoplasma activity of various molecular weights and concentrations of chitosan nanoparticles on tachyzoites of RH strain.

PubMed

Teimouri, Aref; Azami, Sanaz Jafarpour; Keshavarz, Hossein; Esmaeili, Fariba; Alimi, Rasoul; Mavi, Sara Ayazian; Shojaee, Saeedeh

2018-01-01

Natural polysaccharides such as chitosan (CS) are widely used as antimicrobial agents. In recent years, and considering that CS has a strong antimicrobial potential, interest has been focused on antimicrobial activity of chitosan nanoparticles (CS NPs). The main factors affecting the antibacterial activity of chitosan include molecular weight (MW) and concentration. In this regard, the aim of this study was to produce various MWs and concentrations of CS NPs, through the ionic gelation method, and investigate their potential anti-parasitic activity against tachyzoites of Toxoplasma gondii RH strain. The MWs and degree of deacetylation of the CS were characterized using viscometric and acid-base titration methods, respectively. The efficacy of various MWs and concentrations of NPs was assessed by performing in vitro experiments for tachyzoites of T. gondii RH strain, such as MTT assay, scanning electron microscopy, bioassay in mice and PCR. In vivo experiment was carried out in BALB/c mice which were inoculated with tachyzoites of T. gondii RH strain and treated with various MWs of CS NPs. The results of in vitro and in vivo experiments revealed that anti- Toxoplasma activity strengthened as the CS NPs concentration increased and the MW decreased. In vitro experiment showed 100% mortality of tachyzoites at 500 and 1,000 ppm concentrations of low molecular weight (LMW) CS NPs after 180 min and at 2,000 ppm after 120 min. Furthermore, a 100% mortality of tachyzoites was observed at 1,000 and 2,000 ppm concentrations of medium molecular weight (MMW) CS NPs and at 2,000 ppm concentration of high molecular weight (HMW) CS NPs after 180 min. Growth inhibition rates of tachyzoites in peritoneal exudates of mice receiving low, medium and high MWs of CS NPs were found to be 86%, 84% and 79% respectively, compared to those of mice in sulfadiazine treatment group (positive control). Various MWs of CS NPs exhibited great anti- Toxoplasma efficiency against tachyzoites of RH strain, with the greatest efficacy shown by LMW CS NPs in both experiments. It seems that CS NPs can be used as an alternative natural medicine in the treatment of toxoplasmosis.

In vitro efficacy and release study with anti-inflammatory drugs incorporated in adhesive transdermal drug delivery systems.

PubMed

Meyer, Stefanie; Peters, Nils; Mann, Tobias; Wolber, Rainer; Pörtner, Ralf; Nierle, Jens

2014-04-01

The topical application of two different anti-inflammatory extracts incorporated in adhesive transdermal drug delivery systems (TDDSs) was investigated. Therefore, anti-inflammatory properties and percutaneous absorption behavior of adhesive TDDSs were characterized in vitro conducting experiments with a dermatologically relevant human skin model. Anti-inflammatory efficacy against UV irradiation of both TDDSs was determined in vitro with EpiDerm™. The reduction of the release of proinflammatory cytokines by topically applied TDDSs was compared with the reduction during the presence of the specific cyclooxygenase inhibitor diclofenac in the culture medium. A similar anti-inflammatory efficacy of the topically applied TDDSs in comparison with the use of diclofenac in the culture medium should be achieved. Furthermore, percutaneous absorption in efficacy tests was compared with percutaneous absorption in diffusion studies with porcine cadaver skin. Both the topically applied TDDSs showed a significant anti-inflammatory activity. Permeation coefficients through the stratum corneum and the epidermis gained from the release studies on porcine cadaver skin (Magnolia: 2.23·10(-5) cm/h, licorice: 4.68·10(-6) cm/h) were approximately five times lower than the permeation coefficients obtained with the EpiDerm™ skin model (Magnolia: 9.48·10(-5) cm/h, licorice: 24.0·10(-6) cm/h). Therefore, an adjustment of drug doses during experiments with the EpiDerm™ skin model because of weaker skin barrier properties should be considered.

Sustained release effects of berberine-loaded chitosan microspheres on in vitro chondrocyte culture.

PubMed

Zhou, Yan; Liu, Shiqing; Ming, Jianghua; Li, Yaming; Deng, Ming; He, Bin

2017-10-01

The low bioavailability and short biological half-life of berberine chloride (BBR) negatively affect the protective role of this compound against osteoarthritis (OA). The present study was performed to evaluate the effectiveness of sustained BBR release system. Novel BBR-loaded chitosan microspheres (BBR-loaded CMs) were successfully synthesized using an ionic cross-linking method for sustained release. The basic characteristics of the prepared microspheres were subsequently evaluated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) techniques, encapsulation efficiency (EE), and in vitro release experiments. BBR-loaded CMs displayed spherical forms to encapsulate a considerable quantity of BBR (100.8 ± 2.7 mg/g); these microspheres also exhibited an ideal releasing profile. The FT-IR spectra and XRD results revealed that BBR-loaded CMs were successfully synthesized via electrostatic interaction. In vitro experiments further showed that BBR-loaded CMs significantly inhibited sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, and led to increasing mitochondrial membrane potential and maintaining the nuclear morphology. BBR-loaded CMs exerted markedly higher anti-apoptotic activity in the treatment of OA, and markedly inhibited the protein expression levels of caspase-3, a disintegrin, and metalloproteinase with thrombospondin motifs (ADAMTS)-5 and matrix metalloproteinase (MMP)-13 induced by SNP in rat articular chondrocytes, compared with free BBR at equivalent concentration. Therefore, novel BBR-loaded CMs may offer potential for application in the treatment of OA.

In vitro and in vivo comparison of a tailored magnetic particle imaging blood pool tracer with Resovist.

PubMed

Kaul, Michael Gerhard; Mummert, Tobias; Jung, Caroline; Salamon, Johannes; Khandhar, Amit P; Ferguson, R Matthew; Kemp, Scott J; Ittrich, Harald; Krishnan, Kannan M; Adam, Gerhard; Knopp, Tobias

2017-05-07

Optimizing tracers for individual imaging techniques is an active field of research. The purpose of this study was to perform in vitro and in vivo magnetic particle imaging (MPI) measurements using a new monodisperse and size-optimized tracer, LS-008, and to compare it with the performance of Resovist, the standard MPI tracer. Magnetic particle spectroscopy (MPS) and in vitro MPI measurements were performed in concerns of concentration and amount of tracer in a phantom. In vivo studies were carried out in healthy FVB mice. The first group (n  =  3) received 60 µl LS-008 (87 mM) and the second (n  =  3) diluted Resovist of the same concentration and volume. Tracer injections were performed with a syringe pump during a dynamic MPI scan. For anatomic referencing MRI was applied beforehand of the MPI measurements. Summing up MPS examinations and in vitro MPI experiments, LS-008 showed better sensitivity and spatial resolution than Resovist. In vivo both tracers can visualize the propagation of the bolus through the inferior vena cava. MPI with LS-008 did show less temporal fluctuation artifacts and the pulsation of blood due to respiratory and cardiac cycle was detectable. With LS-008 the aorta was distinguishable from the caval vein while with Resovist this failed. A liver vessel and a vessel structure leading cranially could only be observed with LS-008 and not with Resovist. Beside these structural advantages both tracers showed very different blood half-life. For LS-008 we found 88 min. Resovist did show a fast liver accumulation and a half-life of 13 min. Only with LS-008 the perfusion fraction in liver and kidney was measureable. MPI for angiography can be significantly improved by applying more effective tracers. LS-008 shows a clear improvement concerning the delineation while resolving a larger number of vessels in comparison to Resovist. Therefore, in aspects of quality and quantity LS-008 is clearly favorable for angiographic and perfusion studies.

In vitro and in vivo comparison of a tailored magnetic particle imaging blood pool tracer with Resovist

NASA Astrophysics Data System (ADS)

Kaul, Michael Gerhard; Mummert, Tobias; Jung, Caroline; Salamon, Johannes; Khandhar, Amit P.; Ferguson, R. Matthew; Kemp, Scott J.; Ittrich, Harald; Krishnan, Kannan M.; Adam, Gerhard; Knopp, Tobias

2017-05-01

Optimizing tracers for individual imaging techniques is an active field of research. The purpose of this study was to perform in vitro and in vivo magnetic particle imaging (MPI) measurements using a new monodisperse and size-optimized tracer, LS-008, and to compare it with the performance of Resovist, the standard MPI tracer. Magnetic particle spectroscopy (MPS) and in vitro MPI measurements were performed in concerns of concentration and amount of tracer in a phantom. In vivo studies were carried out in healthy FVB mice. The first group (n  =  3) received 60 µl LS-008 (87 mM) and the second (n  =  3) diluted Resovist of the same concentration and volume. Tracer injections were performed with a syringe pump during a dynamic MPI scan. For anatomic referencing MRI was applied beforehand of the MPI measurements. Summing up MPS examinations and in vitro MPI experiments, LS-008 showed better sensitivity and spatial resolution than Resovist. In vivo both tracers can visualize the propagation of the bolus through the inferior vena cava. MPI with LS-008 did show less temporal fluctuation artifacts and the pulsation of blood due to respiratory and cardiac cycle was detectable. With LS-008 the aorta was distinguishable from the caval vein while with Resovist this failed. A liver vessel and a vessel structure leading cranially could only be observed with LS-008 and not with Resovist. Beside these structural advantages both tracers showed very different blood half-life. For LS-008 we found 88 min. Resovist did show a fast liver accumulation and a half-life of 13 min. Only with LS-008 the perfusion fraction in liver and kidney was measureable. MPI for angiography can be significantly improved by applying more effective tracers. LS-008 shows a clear improvement concerning the delineation while resolving a larger number of vessels in comparison to Resovist. Therefore, in aspects of quality and quantity LS-008 is clearly favorable for angiographic and perfusion studies.

In vitro microbial protein synthesis, ruminal degradation and post-ruminal digestibility of crude protein of dairy rations containing Quebracho tannin extract.

PubMed

Castro-Montoya, J; Westreicher-Kristen, E; Henke, A; Diaby, M; Susenbeth, A; Dickhoefer, U

2018-02-01

This study evaluated the effects of Quebracho tannin extract (QTE) on in vitro ruminal fermentation, chemical composition of rumen microbes, ruminal degradation and intestinal digestibility of crude protein (iCPd). Three treatments were tested, the control (basal diet without QTE), the basal diet with 15 g QTE/kg dry matter (DM) and the basal diet with 30 g QTE/kg DM. The basal diet contained (g/kg DM): 339 grass silage, 317 maize silage and 344 concentrate. In vitro gas production kinetic was determined using the Hohenheim gas test (Experiment 1). The Ankom RF technique, a batch system with automatic gas pressure recordings, was used to determine in vitro production of short-chain fatty acids (SCFA) and ammonia-nitrogen concentration (NH 3 -N), as well as nitrogen and purine bases content in liquid-associated microbes (LAM) and in a residue of undegraded feed and solid-associated microbes (Feed+SAM) (Experiment 2). Ruminal degradation and iCPd were determined using the nylon bag technique and the mobile nylon bag technique, respectively (Experiment 3). Gas production (Experiment 1), total SCFA and NH 3 -N (Experiment 2) decreased with increasing QTE levels. Microbial mass and composition of LAM were not affected by QTE, but total mass of Feed+SAM linearly increased, likely due to decreased substrate degradation with increasing QTE levels. The total amount of N in microbial mass and undegraded feed after the in vitro incubation increased with increasing QTE levels, suggesting a potential greater N flow from the rumen to the duodenum. In contrast to in vivo studies with the same QTE, no effects were detected on ruminal effective degradability and iCPd, when using the nylon bag techniques. Based on the in vitro procedures, QTE increased the supply of N post-rumen; however, some evidence of a decreased fibre degradation were also observed. Therefore, the benefit of adding QTE to diets of cattle is still questionable. © 2017 Blackwell Verlag GmbH.

Biphasic Dose Response in Low Level Light Therapy – An Update

PubMed Central

Huang, Ying-Ying; Sharma, Sulbha K; Carroll, James; Hamblin, Michael R

2011-01-01

Low-level laser (light) therapy (LLLT) has been known since 1967 but still remains controversial due to incomplete understanding of the basic mechanisms and the selection of inappropriate dosimetric parameters that led to negative studies. The biphasic dose-response or Arndt-Schulz curve in LLLT has been shown both in vitro studies and in animal experiments. This review will provide an update to our previous (Huang et al. 2009) coverage of this topic. In vitro mediators of LLLT such as adenosine triphosphate (ATP) and mitochondrial membrane potential show biphasic patterns, while others such as mitochondrial reactive oxygen species show a triphasic dose-response with two distinct peaks. The Janus nature of reactive oxygen species (ROS) that may act as a beneficial signaling molecule at low concentrations and a harmful cytotoxic agent at high concentrations, may partly explain the observed responses in vivo. Transcranial LLLT for traumatic brain injury (TBI) in mice shows a distinct biphasic pattern with peaks in beneficial neurological effects observed when the number of treatments is varied, and when the energy density of an individual treatment is varied. Further understanding of the extent to which biphasic dose responses apply in LLLT will be necessary to optimize clinical treatments. PMID:22461763

Silk fibroin nanoparticles prepared by electrospray as controlled release carriers of cisplatin.

PubMed

Qu, Jing; Liu, Yu; Yu, Yanni; Li, Jing; Luo, Jingwan; Li, Mingzhong

2014-11-01

To maintain the anti-tumor activity of cis-dichlorodiamminoplatinum (CDDP) while avoiding its cytotoxicity and negative influence on normal tissue, CDDP-loaded silk fibroin nanoparticles approximately 59 nm in diameter were successfully prepared by electrospray without using organic solvent. CDDP was incorporated into nanoparticles through metal-polymer coordination bond exchange. In vitro release tests showed that the cisplatin in the nanoparticles could be slowly and sustainably released for more than 15 days. In vitro anti-cancer experiments and intracellular Pt content testing indicated that CDDP-loaded silk fibroin nanoparticles were easily internalized by A549 lung cancer cells, transferring CDDP into cancer cells and then triggering their apoptosis. In contrast, the particles were not easily internalized by L929 mouse fibroblast cells and hence showed weaker cell growth inhibition. The CDDP-loaded silk fibroin nanoparticles showed sustained and efficient killing of tumor cells but weaker inhibition of normal cells. In general, this study provides not only a novel method for preparing CDDP-loaded silk fibroin nanoparticles but also a new carrier system for clinical therapeutic drugs against lung cancers and other tumors. Copyright © 2014 Elsevier B.V. All rights reserved.

Positive effects of Forskolin (stimulator of lipolysis) treatment on cryosurvival of in vitro matured porcine oocytes.

PubMed

Fu, Xiang-Wei; Wu, Guo-Quan; Li, Jun-Jie; Hou, Yun-Peng; Zhou, Guang-Bin; Lun-Suo; Wang, Yan-Ping; Zhu, Shi-En

2011-01-15

In order to examine its effect on oocyte lipid content and cryosurvival, Forskolin was added to the medium for in vitro maturation of porcine oocytes. Treatments were control (IVM without Forskolin during the 42 h incubation period), addition of 10 μM Forskolin for the entire 42 h (0-42) and addition of 10 μM Forskolin between 24 and 42 h only (24-42). In Experiment 1, treatments did not differ significantly in cleavage rate, but the blastocyst formation rate was lower in the 0-42 group than for control and 24-42 group oocytes (17, 32 and 40%, respectively; P < 0.05). It was shown in Experiment 2 that Forskolin treatment from 0-42 h and from 24-42 h significantly reduced lipid content of oocytes compared to that of control cells (65 and 99 vs. 140 μm(2) intensity of fluorescence, respectively; P < 0.05). In Experiment 3, the percentage of oocyte survival after cryopreservation and thawing was significantly higher in both Forskolin treatment groups than in control oocytes (72% for 0-42, 65% for 24-42 and 52% for control; P < 0.05). However, Forskolin treatment did not increase cleavage rates of vitrified in vitro matured porcine oocytes (Control group 28%, 0-42 h group 0%, 24-42 h group 26.67%). Addition of Forskolin affected the nuclear maturation of porcine oocytes. The percentage of PBE (polar body extrusion) were significantly reduced in the 0-42 h group (0-42 h group 42.00 ± 2.08 vs. Control group 79.70 ± 2.82 and 24-42 h group 70.60 ± 2.83; P < 0.05). The 24-42 h group showed similar nuclear status to that of the Control group. We propose that delipation engendered by incubation with 10 μM Forskolin during 24-42 hours of maturation increased cryosurvival of in vitro-maturated porcine oocytes and that attendant chemical lipolysis did not impair their further development as it may have done in oocytes incubated with Forskolin for the full 42 h. Copyright © 2011 Elsevier Inc. All rights reserved.

M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

PubMed

De Angelis, Federica; Marinelli, Sara; Fioretti, Bernard; Catacuzzeno, Luigi; Franciolini, Fabio; Pavone, Flaminia; Tata, Ada Maria

2014-06-01

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments. © 2013 Wiley Periodicals, Inc.

Ultrastructure changes in buffalo (Bubalus bubalis) oocytes before and after maturation in vitro with sericin.

PubMed

Gustina, Sri; Hasbi, Hasbi; Karja, Ni Wayan Kurniani; Setiadi, Mohamad Agus; Supriatna, Iman

2017-12-01

The aim of this research was to identify the changes in the cytoplasmic ultrastructure of immature and matured oocytes in buffalo (Bubalus bubalis). Oocytes were matured in vitro in tissue culture medium-199 with and without sericin, and then analyzed by light and transmission electron microscopy. The experiment result showed that the nuclear maturation rate of buffalo oocytes was significantly higher in the presence of sericin (80.6%) than without sericin (68.1%) (P < 0.05). The immature oocytes were characterized by cortical granule clusters in the ooplasm and the absence of perivitelline space (PVS). In contrast, the oocytes matured either with or without sericin showed the formation of PVS, erected microvilli, the migration of cortical granules to the cytoplasmic periphery, and the clear appearance of the mitochondria and vesicle in the oolemma. Interestingly, matured oocytes with sericin have smaller cortical granules than do immature oocytes (P < 0.05). In conclusion, supplementation of 0.05% sericin in the maturation medium can enhance the maturation rate of buffalo oocytes. Several cytoplasmic ultrastructures were relocated and modulated during the in vitro maturation process of buffalo oocytes: PVS development, cortical granules migration to periphery, and mitochondria and vesicles in the cortical region. The ultrastructure was similar between the groups with and without sericin. © 2017 Japanese Society of Animal Science.

Radiosensitization of high-Z compounds by medium-energy 160 kV vs. high-energy 6 MV X-rays for radiation therapy: Theoretical, in vitro and in vivo studies of platinum compounds activating glioma F98 cancer cells

NASA Astrophysics Data System (ADS)

Lim, S.; Pradhan, A.; Nahar, S.; Montenegro, M.; Barth, R.; Nakkula, R.; Turro, C.

2013-03-01

Energy dependence of X-ray irradiation of high-Z compounds for enhanced radiosensitization is explored thoeretically and via in vitro and in vivo experiments. The cell killing ability of medium-energy X-rays from 160 kV source are found to be more effective than 6 MV X-rays in activating high-Z contrast agents. Results are presented for a newly synthesized Pt compound, Pyridine Terpyridine Pt(II) Nitrate ([Pt(typ)(py)]) and carboplatin in treating F98 rat glioma. In-vitro results show considerable reduction in cell viability for radiosensitized cells irradiated with a 160 kV irradiator. Cells treated with 6 MV LINAC radiation find little variation with radiation dose. Maximum dose enhancement factors (DEFs) and minimum cancer cell survival fractions correspond to 50-200 keV range, and fall rapidly at higher energies. Theoretical calculations of photoelectric absorption vis-a-vis total scattering demonstrates this energy dependence. However, in vivo studies of rats treated with [Pt(tpy)(py)] had a severe negative neurotoxic response, confirmed by histopathological analysis. But subsequent in vivo studies using carboplatin showed very positive results in the treatment of F98 glioma bearing rats and potential clinical radiation therapy.

Design, characterization, and in vitro cellular inhibition and uptake of optimized genistein-loaded NLC for the prevention of posterior capsular opacification using response surface methodology.

PubMed

Zhang, Wenji; Li, Xuedong; Ye, Tiantian; Chen, Fen; Sun, Xiao; Kong, Jun; Yang, Xinggang; Pan, Weisan; Li, Sanming

2013-09-15

This study was to design an innovative nanostructured lipid carrier (NLC) for drug delivery of genistein applied after cataract surgery for the prevention of posterior capsular opacification. NLC loaded with genistein (GEN-NLC) was produced with Compritol 888 ATO, Gelucire 44/14 and Miglyol 812N, stabilized by Solutol(®) HS15 by melt emulsification method. A 2(4) central composite design of 4 independent variables was performed for optimization. Effects of drug concentration, Gelucire 44/14 concentration in total solid lipid, liquid lipid concentration, and surfactant concentration on the mean particle size, polydispersity index, zeta potential and encapsulation efficiency were investigated. Analysis of variance (ANOVA) statistical test was used to assess the optimization. The optimized GEN-NLC showed a homogeneous particle size of 90.16 nm (with PI=0.33) of negatively charged surface (-25.08 mv) and high encapsulation efficiency (91.14%). Particle morphology assessed by TEM revealed a spherical shape. DSC analyses confirmed that GEN was mostly entrapped in amorphous state. In vitro release experiments indicated a prolonged and controlled genistein release for 72 h. In vitro growth inhibition assay showed an effective growth inhibition of GEN-NLCs on human lens epithelial cells (HLECs). Preliminary cellular uptake test proved a enhanced penetration of genistein into HLECs when delivered in NLC. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of low-molecular-weight-chitosan on the adenine-induced chronic renal failure rats in vitro and in vivo

NASA Astrophysics Data System (ADS)

Zhi, Xuan; Han, Baoqin; Sui, Xianxian; Hu, Rui; Liu, Wanshun

2015-02-01

The effects of low-molecular-weight-chitosan (LMWC) on chronic renal failure (CRF) rats induced by adenine were investigated in vivo and in vitro. Chitosan were hydrolyzed using chitosanase at pH 6-7 and 37° for 24 h to obtain LMWC. In vitro, the effect of LMWC on the proliferation of renal tubular epithelial cells (RTEC) showed that it had no cytotoxic effect and could promote cell growth. For the in vivo experiment, chronic renal failure rats induced by adenine were randomly divided into control group, Niaoduqing group, and high-, medium- and low-dose LMWC groups. For each group, we detected serum creatinine (SCR), blood urea nitrogen (BUN), and total superoxide dismutase (T-SOD), glutathione oxidase (GSH-Px) activities of renal tissue, and obtained the ratio of kidney weight/body weight, pathological changes of kidney. The levels of serum SCR, BUN were higher in the adenine-induced rats than those in the control group, indicating that the rat chronic renal failure model worked successfully. The results after treatment showed that LMWC could reduce the SCR and BUN levels and enhance the activities/levels of T-SOD and GSH-PX in kidney compared to control group. Histopathological examination revealed that adenine-induced renal alterations were restored by LMWC at three tested dosages, especially at the low dosage of 100 mg kg-1 d-1.

Influence of cell culture medium composition on in vitro dissolution behavior of a fluoride-containing bioactive glass.

PubMed

Shah, Furqan A; Brauer, Delia S; Wilson, Rory M; Hill, Robert G; Hing, Karin A

2014-03-01

Bioactive glasses are used clinically for bone regeneration, and their bioactivity and cell compatibility are often characterized in vitro, using physiologically relevant test solutions. The aim of this study was to show the influence of varying medium characteristics (pH, composition, presence of proteins) on glass dissolution and apatite formation. The dissolution behavior of a fluoride-containing bioactive glass (BG) was investigated over a period of one week in Eagle's Minimal Essential Medium with Earle's Salts (MEM), supplemented with either, (a) acetate buffer, (b) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, (c) HEPES + carbonate, or (d) HEPES + carbonate + fetal bovine serum. Results show pronounced differences in pH, ion release, and apatite formation over 1 week: Despite its acidic pH (pH 5.8 after BG immersion, as compared to pH 7.4-8.3 for HEPES-containing media), apatite formation was fastest in acetate buffered (HEPES-free) MEM. Presence of carbonate resulted in formation of calcite (calcium carbonate). Presence of serum proteins, on the other hand, delayed apatite formation significantly. These results confirm that the composition and properties of a tissue culture medium are important factors during in vitro experiments and need to be taken into consideration when interpreting results from dissolution or cell culture studies. Copyright © 2013 Wiley Periodicals, Inc.

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus.

PubMed

Qi, Xiaole; Gao, Xiang; Lu, Zhen; Zhang, Lizhou; Wang, Yongqiang; Gao, Li; Gao, Yulong; Li, Kai; Gao, Honglei; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Wang, Xiaomei

2016-07-01

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

Bacterial cell-free expression technology to in vitro systems engineering and optimization.

PubMed

Caschera, Filippo

2017-06-01

Cell-free expression system is a technology for the synthesis of proteins in vitro . The system is a platform for several bioengineering projects, e.g. cell-free metabolic engineering, evolutionary design of experiments, and synthetic minimal cell construction. Bacterial cell-free protein synthesis system (CFPS) is a robust tool for synthetic biology. The bacteria lysate, the DNA, and the energy module, which are the three optimized sub-systems for in vitro protein synthesis, compose the integrated system. Currently, an optimized E. coli cell-free expression system can produce up to ∼2.3 mg/mL of a fluorescent reporter protein. Herein, I will describe the features of ATP-regeneration systems for in vitro protein synthesis, and I will present a machine-learning experiment for optimizing the protein yield of E. coli cell-free protein synthesis systems. Moreover, I will introduce experiments on the synthesis of a minimal cell using liposomes as dynamic containers, and E. coli cell-free expression system as biochemical platform for metabolism and gene expression. CFPS can be further integrated with other technologies for novel applications in environmental, medical and material science.

Evolution in vitro: analysis of a lineage of ribozymes

NASA Technical Reports Server (NTRS)

Lehman, N.; Joyce, G. F.

1993-01-01

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

Risk of bowel obstruction during in vitro fertilization treatment of patients with deep infiltrating endometriosis.

PubMed

Seyer-Hansen, Mikkel; Egekvist, Anne; Forman, Axel; Riiskjaer, Mads

2018-01-01

Women with endometriosis often experience pain and infertility. Medical treatment interferes with the possibility of attaining pregnancy. For infertile women with endometriosis, surgery is a possible treatment, but with advanced disease there is an increased risk of serious complications. With only limited pain, women will often be referred for in vitro fertilization treatment instead. The disease is estrogen-dependent and during in vitro fertilization treatment the women could theoretically experience worsening of their symptoms. The study is a retrospective cohort study of 76 women with bowel endometriosis who were treated conservatively and underwent in vitro fertilization treatment. Nine (11.8%) of the women experienced severe worsening of their bowel-related symptoms, including two patients presenting with colon ileus. One additional woman had no previous diagnosis of endometriosis before she presented with subocclusion of the bowel during in vitro fertilization. In all cases the in vitro fertilization treatment was stopped. Our study revealed that bowel endometriosis increases the risk of complications during in vitro fertilization treatment. This is in contrast to several publications. However, our study population is different due to the fact that none of these women had previous operations for bowel endometriosis. In all, 88% of the women completed fertility treatment without need for surgery. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Bioactive saponins from the fruits of Aesculus pavia L.

PubMed

Sun, Zhen-Liang; Zhang, Ming; Wu, Ying; Wan, Ai-Hong; Zhang, Rong

2011-10-01

Continued chemical investigation on the fruits of Aesculus pavia L. resulted in theisolation and identification of two new oleanolic acid saponins, namely vaccaroside A (1) andvaccaroside B (2). The isolated furostanol saponins were evaluated for cytotoxic activity againsthuman normal amniotic and human lung carcinoma cell lines using neutral red and MTT assays.In vitro experiments showed significant cytotoxicity in a dose dependent manner with IC₅₀ valuesin the range of 27.80-79.02 μM. Copyright © 2011 Elsevier B.V. All rights reserved.

Investigation of T-2 Mycotoxin-Induced Cytotoxicity in vitro and Protective Effects of Flavonoid Compounds

DTIC Science & Technology

1986-01-01

Quercetin , a flavonoid compound was able to decrease the effect of T-2 toxin when the drug was added within an hour of mixing the T-2 toxin with the...were examined microscopically using a Neubauer hemocytometer and viability of at least 200 cells was deter- mined. Quercetin or other flavonold... quercetin and additional OMSO had a cytotoxic effect on the thymocytes. RESULTS Figure 1 shows the results of 8 separate experiments performed at 2 week

Antiprotozoal Constituents from Annona cherimola Miller, a Plant Used in Mexican Traditional Medicine for the Treatment of Diarrhea and Dysentery

PubMed Central

Calzada, Fernando; Correa-Basurto, Jose; Barbosa, Elizabeth; Mendez-Luna, David; Yepez-Mulia, Lilian

2017-01-01

Background: Annona cherimola Miller (Annonaceae) is a medicinal plant frequently recommended in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. Objective: This work was undertaken to obtain information that support the traditional use of A. cherimola, on pharmacological basis using in vitro and computational experiments. Material and Methods: Bioassay-guided fractionation of the ethanol extract of the leaves of A. cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin, and rutin. Results: The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different than metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase (PFOR). Conclusion: Considering that PFOR is a target of metronidazole; kaempferol may be a lead compound for the development of novel antiprotozoal agent. Also, these findings give support to the use of A. cherimola in the traditional medicine from México for the treatment of diarrhea and dysentery. SUMMARY Bioassay-guided fractionation of the ethanol extract of the leaves of Annona cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin and rutin. The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different that metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase. Abbreviations used: PFOR:Pyruvate:ferredoxin oxidoreductase, G: lamblia: Giardia lamblia, E: histolytica: Entamoeba histolytica PMID:28216899

In vitro antiplasmodial activity and prophylactic potentials of extract and fractions of Trema orientalis (Linn.) stem bark.

PubMed

Olanlokun, John Oludele; David, Oluwole Moses; Afolayan, Anthony Jide

2017-08-15

Trema orientalis (T. orientalis Linn) has been used in the management of malaria in the western part of Nigeria and despite its application in ethnomedicine, there is dearth of scientific evidence to justify the acclaimed prophylactic antimalarial usage of the plant. The aim of this study is to assess the in vitro antiplasmodial cell-free assay and chemopreventive efficacy of the methanol extract of the stem bark of T. orientalis and its fractions as a prophylactic regimen for malaria prevention. Also, the antimicrobial activities of the extract and the fractions were investigated. Vacuum liquid chromatography was used to obtain dichloromethane, ethylacetate and methanol fractions from the methanol extract of T. orientalis. The fractions were tested for their prophylactic and cell-free antimalarial activity using murine models and β-hematin formation assay respectively. Disc diffusion method was used to determine the antibacterial activity of the extract and its fractions against both Gram-positive and Gram-negative bacteria. In the prophylactic experiment, dichloromethane (DCMF), methanol fraction (MF) and extract (ME) (in this order) showed significant chemopreventive effects against P. berghei invasion of the red blood cells when compared with both Sulfadoxine-Pyrimethamine (SP) and untreated controls. Results of the in vitro study showed that the DCMF had the highest effect in preventing the formation of β-hematin when compared with other fractions. The DCMF also had the highest percentage inhibition of β-hematin formation when compared with chloroquine. The extract and fractions showed a concentration dependent antibacterial activity. Methanol extract had a pronounced inhibitory effect on Enterobacter cloaca ATCC 13047 and Enterococcus faecalis ATCC 29212. Serratia mercescens ATCC 9986 and Pseudomonas aeruginosa ATCC 19582 were the most susceptible bacteria. The results obtained showed that both extract and fractions of T. orientalis possessed antiplasmodial and antimicrobial activity.

The Effect of Micro-Gravity on in vitro Calcification

NASA Technical Reports Server (NTRS)

Boskey; Stiner; Binderman; Mendelsohn; Doty, S. B.

1997-01-01

The experiment focuses on mineral deposition or calcification of cartilage. The experiments were used to compare the mineral formed in the microgravity of space with that formed on earth. Results of these experiments were anticipated to provide direct insight into how calcification in cartridge and bone may be controlled in space. In the C-2 experiment (STS 66), we found that mineralization started later in the cartridges (both on the ground and in hypo-gravity) than in plastic, and that mineralization appeared to be retarded in hypo-gravity. The flight experiments also showed that the cells differentiated normally, but more slowly than the ground controls, and that the matrix produced was not different from that made on the ground. The purpose of the C-5 experiment was to confirm these findings. The C-5 experiment was flown on STS-72. Because of a computer problem, cells received no gases and no nutrition. The C-7 was flown on STS-77. Ground controls were repeated a week later, however, because there was a problem with the temperature control during the flight, the concurrent ground controls were performed at a different temperature. Despite these problems, the results of the C-2 experiment were confirmed. The cells in the flight cultures did not mature, formed few cartilage nodules, and showed no evidence of mineral deposition up to a culture age of 28 days. Ground controls showed the presence of mineral (based on chemical, spectroscopic, and histochemical analyses) by 21 days. The mineral in these cultures was analogous to that found in calcifying cartilage of young chicks.

Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability.

PubMed

del Olmo, D; Parrilla, I; Gil, M A; Maside, C; Tarantini, T; Angel, M A; Roca, J; Martinez, E A; Vazquez, J M

2013-09-01

The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies. Copyright © 2013 Elsevier Inc. All rights reserved.

An experiment to study the effects of space flight cells of mesenchymal origin in the new model 3D-graft in vitro

NASA Astrophysics Data System (ADS)

Volova, Larissa

One of the major health problems of the astronauts are disorders of the musculoskeletal system, which determines the relevance of studies of the effect of space flight factors on osteoblastic and hondroblastic cells in vitro. An experiment to study the viability and proliferative activity of cells of mesenchymal origin on culture: chondroblasts and dermal fibroblasts was performed on SC "BION -M" No. 1 with scientific equipment " BIOKONT -B ." To study the effect of space flight conditions in vitro at the cellular level has developed a new model with 3D- graft as allogeneic demineralized spongiosa obtained on technology Lioplast ®. For space and simultaneous experiments in the laboratory of the Institute of Experimental Medicine and Biotechnology Samara State Medical University were obtained from the cell culture of hyaline cartilage and human skin, which have previously been grown, and then identified by morphological and immunohistochemical methods. In the experiment, they were seeded on the porous 3D- graft (controlled by means of scanning electron and confocal microscopy) and cultured in full growth medium. After completion of the flight of spacecraft "BION -M" No. 1 conducted studies of biological objects using a scanning electron microscope (JEOL JSM-6390A Analysis Station, Japan), confocal microscopy and LDH - test. According to the results of the experiment revealed that after a 30- day flight of the cells not only retained vitality, but also during the flight actively proliferate, and their number has increased by almost 8 times. In synchronous experiment, all the cells died by this date. The experimentally confirmed the adequacy of the proposed model 3D- graft in studying the effect of space flight on the morphological and functional characteristics of cells in vitro.

Reproducibility and Consistency of In Vitro Nucleosome Reconstitutions Demonstrated by Invitrosome Isolation and Sequencing

PubMed Central

Kempton, Colton E.; Heninger, Justin R.; Johnson, Steven M.

2014-01-01

Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems. PMID:25093869

[Effect of rat intestinal flora on in vitro metabolic transformation of pumiloside].

PubMed

Fang, Hui; Li, Meng-Xuan; Li, Hai-Bo; Liu, Wen-Jun; Meng, Zhao-Qing; Huang, Wen-Zhe; Wang, Zhen-Zhong; Xiao, Wei

2016-05-01

To study the metabolic transformation of pumiloside by rat intestinal flora in vitro and identify its metabolites. Pumiloside was incubated in the rat intestinal flora in vitro. HPLC was used to monitor the metabolic process, and HPLC-Q-TOF-MS was used to identify the structures of biotransformation products. In vitro, pumiloside was easily metabolized by rat intestinal flora, and with the prolongation of metabolic time, pumiloside was transformed into several metabolites. Three metabolites were initially identified in this experiment. The study indicated that pumiloside could be extensively metabolized in the rat intestinal flora in vitro. Copyright© by the Chinese Pharmaceutical Association.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

PubMed

Bozue, Joel A; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K; Toothman, Ronald G; Dankmeyer, Jennifer L; Klimko, Christopher P; Wilhelmsen, Catherine L; Raymond, Jolynn W; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice

PubMed Central

Bozue, Joel A.; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K.; Toothman, Ronald G.; Dankmeyer, Jennifer L.; Klimko, Christopher P.; Wilhelmsen, Catherine L.; Raymond, Jolynn W.; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders. PMID:26955620

Syntheses, receptor bindings, in vitro and in vivo stabilities and biodistributions of DOTA-neurotensin(8-13) derivatives containing β-amino acid residues - a lesson about the importance of animal experiments.

PubMed

Sparr, Christof; Purkayastha, Nirupam; Yoshinari, Tomohiro; Seebach, Dieter; Maschauer, Simone; Prante, Olaf; Hübner, Harald; Gmeiner, Peter; Kolesinska, Beata; Cescato, Renzo; Waser, Beatrice; Reubi, Jean Claude

2013-12-01

Neurotensin(8-13) (NTS(8-13)) analogs with C- and/or N-terminal β-amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1-6). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, 6a, into a crystallographically identified receptor NTSR1 (Fig.1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell-membrane homogenates, while, with NTSR1-exhibiting cancer tissues, affinities in the single-digit nanomolar range can be observed (Table 2). Most of the β-amino acid-containing NTS(8-13) analogs (Table 1 and Fig.2), including the (68) Ga complexes of the DOTA-substituted ones (6; Figs.2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two (68) Ga complexes (of 6a and 6b) in HT29 tumor-bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET-imaging experiments with the tumor-bearing mice (Fig.6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10-15 min) of the two (68) Ga complexes shows that they are rapidly cleaved in the animals (Fig.5). Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

The Effect of the Gambir (Uncaria gambir (Hunt.) Roxb.) Leaves Waste and White Turmeric (Curcuma zedoaria) for The Productivity, Antioxidant Content and Mastitis Condition of The Fries Holland Dairy Cows

NASA Astrophysics Data System (ADS)

Nurdin, E.; Fitrimawati

2018-02-01

The aim of this research was to evaluate the effect of the gambir leaves waste and white turmeric on the productivity, antioxidant content and mastitis condition of dairy cows. The treatments were A. 0% gambir waste + 0.02% white turmeric; B. 20% of gambir waste + 0.02% white turmeric; C. 30% gambir waste + 0.02% white turmeric; D. 40% gambir waste + 0.02% white turmeric; and E. 50% gambir waste + 0.02% white turmeric. The results of in vitro experiment showed that the optimum ecological conditions rumen pH were ranging from 6.66 to 7.59, NH3 concentrations were ranging from 23.42 to 31.54 nM and VFA concentrations were ranging from 66.15 to 119.82 mM. All treatments gave a real impact on the rumen ecology of dairy cows FH. In-vivo experiment for the best treatment from in-vitro experiment (30% gambir waste + 0.02% white turmeric) showed that similarly, the effect of administration of gambir leaves waste and white turmeric on dairy cattle FH, antioxidant activity affect the increase of milk production (P<0.05) from 9.75 liters to 11.52 liters (18.15%), increase the antioxidant content of milk (P<0.05) from 10.98 to 13.89 (26.50%) and a decline of the mastitis (P<0.05) from positive two to positive one. Gambir leaves decoction waste at the amount of 30% combined with white turmeric gave the optimal condition of rumen ecology. It was also increased milk production by 18.15%, the antioxidant content of milk by 26.50%, and decline mastitis.

Effect of an oxygenating agent on oral bacteria in vitro and on dental plaque composition in healthy young adults.

PubMed

Fernandez y Mostajo, Mercedes; van der Reijden, Wil A; Buijs, Mark J; Beertsen, Wouter; Van der Weijden, Fridus; Crielaard, Wim; Zaura, Egija

2014-01-01

Oral bacteria live in symbiosis with the host. Therefore, when mouthwashes are indicated, selective inhibition of taxa contributing to disease is preferred instead of broad-spectrum antimicrobials. The potential selectivity of an oxygenating mouthwash, Ardox-X® (AX), has not been assessed. The aim of this study was to determine the antimicrobial potential of AX and the effects of a twice-daily oral rinse on dental plaque composition. In vitro, 16 oral bacterial strains were tested using agar diffusion susceptibility, minimum inhibitory and minimum bactericidal concentration tests. A pilot clinical study was performed with 25 healthy volunteers. Clinical assessments and microbiological sampling of supragingival plaque were performed at 1 month before the experiment (Pre-exp), at the start of the experiment (Baseline) and after the one-week experimental period (Post-exp). During the experiment individuals used AX mouthwash twice daily in absence of other oral hygiene measures. The microbiological composition of plaque was assessed by 16S rRNA gene amplicon sequencing. AX showed high inter-species variation in microbial growth inhibition. The tested Prevotella strains and Fusobacterium nucleatum showed the highest sensitivity, while streptococci and Lactobacillus acidophilus were most resistant to AX. Plaque scores at Pre-exp and Baseline visits did not differ significantly (p = 0.193), nor did the microbial composition of plaque. During a period of 7-days non-brushing but twice daily rinsing plaque scores increased from 2.21 (0.31) at Baseline to 2.43 (0.39) Post-exp. A significant microbial shift in composition was observed: genus Streptococcus and Veillonella increased while Corynebacterium, Haemophilus, Leptotrichia, Cardiobacterium and Capnocytophaga decreased (p ≤ 0.001). AX has the potential for selective inhibition of oral bacteria. The shift in oral microbiome after 1 week of rinsing deserves further research.

A homeopathic remedy from arnica, marigold, St. John's wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts.

PubMed

Hostanska, Katarina; Rostock, Matthias; Melzer, Joerg; Baumgartner, Stephan; Saller, Reinhard

2012-07-18

Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2), its succussed hydroalcoholic solvent (0712-1) and unsuccussed solvent (0712-3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined "wound field". All assays were performed in three independent controlled experiments. None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712-1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712-3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. Results of this study showed that the low potency homeopathic remedy (0712-2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.

Gravitational physiology of human immune cells: a review of in vivo, ex vivo and in vitro studies

NASA Technical Reports Server (NTRS)

Cogoli, A.

1996-01-01

The study of the function of immune cells in microgravity has been studied for more than 20 years in several laboratories. It is clear today that the immune system is depressed in more than 50% of the astronauts during and after space flight and that the activation of T lymphocytes by mitogens in vitro changes dramatically. This article gives an overview of the gravitational studies conducted by our laboratory in Spacelab, in MIR station, in sounding rockets and on the ground in the clinostat and the centrifuge. Three experimental approaches are followed in our work: (i) Ex vivo studies are performed with blood samples drawn from astronauts; (ii) in vivo studies are based on the application of seven antigens to the skin of the astronauts; (iii) in vitro studies are carried out with immune cells purified from the blood of healthy donors (not astronauts). The data from our in vivo and ex vivo studies are in agreement with those of other laboratories and show that the immunological function is depressed in the majority of astronauts as a consequence of the stress of space flight rather than by a direct influence of gravity on the cell. Immune depression may become a critical hazard on long duration flights on space stations or to other planets. In vitro experiments show that cultures of free-floating lymphocytes and monocytes undergo a dramatic depression of activation by the mitogen concanavalin A, while activation is more than doubled when the cells are attached to microcarrier beads. Such effects may be attributed to both direct and indirect effects of gravitational unloading on basic biological mechanisms of the cell. While the in vitro data are very important to clarify certain aspects of the biological mechanism of T cells activation, they are not descriptive of the changes of the immunological function of the astronauts.

Gravitational physiology of human immune cells: a review of in vivo, ex vivo and in vitro studies.

PubMed

Cogoli, A

1996-04-01

The study of the function of immune cells in microgravity has been studied for more than 20 years in several laboratories. It is clear today that the immune system is depressed in more than 50% of the astronauts during and after space flight and that the activation of T lymphocytes by mitogens in vitro changes dramatically. This article gives an overview of the gravitational studies conducted by our laboratory in Spacelab, in MIR station, in sounding rockets and on the ground in the clinostat and the centrifuge. Three experimental approaches are followed in our work: (i) Ex vivo studies are performed with blood samples drawn from astronauts; (ii) in vivo studies are based on the application of seven antigens to the skin of the astronauts; (iii) in vitro studies are carried out with immune cells purified from the blood of healthy donors (not astronauts). The data from our in vivo and ex vivo studies are in agreement with those of other laboratories and show that the immunological function is depressed in the majority of astronauts as a consequence of the stress of space flight rather than by a direct influence of gravity on the cell. Immune depression may become a critical hazard on long duration flights on space stations or to other planets. In vitro experiments show that cultures of free-floating lymphocytes and monocytes undergo a dramatic depression of activation by the mitogen concanavalin A, while activation is more than doubled when the cells are attached to microcarrier beads. Such effects may be attributed to both direct and indirect effects of gravitational unloading on basic biological mechanisms of the cell. While the in vitro data are very important to clarify certain aspects of the biological mechanism of T cells activation, they are not descriptive of the changes of the immunological function of the astronauts.

A minimal physical model for crawling cells

NASA Astrophysics Data System (ADS)

Tiribocchi, Adriano; Tjhung, Elsen; Marenduzzo, Davide; Cates, Michael E.

Cell motility in higher organisms (eukaryotes) is fundamental to biological functions such as wound healing or immune response, and is also implicated in diseases such as cancer. For cells crawling on solid surfaces, considerable insights into motility have been gained from experiments replicating such motion in vitro. Such experiments show that crawling uses a combination of actin treadmilling (polymerization), which pushes the front of a cell forward, and myosin-induced stress (contractility), which retracts the rear. We present a simplified physical model of a crawling cell, consisting of a droplet of active polar fluid with contractility throughout, but treadmilling connected to a thin layer near the supporting wall. The model shows a variety of shapes and/or motility regimes, some closely resembling cases seen experimentally. Our work supports the view that cellular motility exploits autonomous physical mechanisms whose operation does not need continuous regulatory effort.

A minimal physical model captures the shapes of crawling cells

NASA Astrophysics Data System (ADS)

Tjhung, E.; Tiribocchi, A.; Marenduzzo, D.; Cates, M. E.

2015-01-01

Cell motility in higher organisms (eukaryotes) is crucial to biological functions ranging from wound healing to immune response, and also implicated in diseases such as cancer. For cells crawling on hard surfaces, significant insights into motility have been gained from experiments replicating such motion in vitro. Such experiments show that crawling uses a combination of actin treadmilling (polymerization), which pushes the front of a cell forward, and myosin-induced stress (contractility), which retracts the rear. Here we present a simplified physical model of a crawling cell, consisting of a droplet of active polar fluid with contractility throughout, but treadmilling connected to a thin layer near the supporting wall. The model shows a variety of shapes and/or motility regimes, some closely resembling cases seen experimentally. Our work strongly supports the view that cellular motility exploits autonomous physical mechanisms whose operation does not need continuous regulatory effort.

Evaluation of cholesterol- treated dromedary camel sperm function by heterologous IVF and AI.

PubMed

Crichton, Elizabeth G; Malo, Clara; Pukazhenthi, Budhan S; Nagy, Peter; Skidmore, Julian A

2016-11-01

Cholesterol (cholesterol-loaded cyclodextrins: CLC) treatment of dromedary camel sperm prior to freezing enhances cryosurvival. The present study first validated the efficacy of a heterologous zona-free goat oocyte assay (n=115 oocytes) to evaluate camel sperm function in vitro (Experiment 1: n=6 bulls), then examined the effects of CLC treatment (1.5mg/mL CLC; CLC+) versus no treatment (0 CLC) of fresh (Experiment 2: n=4 bulls) and frozen-thawed (Experiment 3: n=5 bulls) camel sperm to penetrate, de-condense and form pro-nuclei in in vitro-matured goat oocytes. Finally, the ability of fresh 0 CLC and CLC+ sperm to fertilize in vivo was studied by artificially inseminating super-ovulated females (n=7-9 per treatment) and examining embryo production (Experiment 4: n=4-5 bulls/treatment). Camel spermatozoa penetrated (60%) and formed pro-nuclei (33%) in goat oocytes demonstrating the utility of this heterologous system for assessing sperm function in vitro. For fresh spermatozoa, 0 CLC-treated sperm performed better than their CLC+ counterparts for all parameters measured (P<0.05). In contrast, cryopreservation resulted in a sharp decline in sperm-oocyte interaction in 0 CLC aliquots but remained unaltered in CLC+ aliquots demonstrating a protective effect of cholesterol treatment. There was no difference between treatments in the in vitro fertilizing ability of frozen-thawed sperm or in the numbers of embryos retrieved following AI with fresh 0 CLC or CLC+ sperm. We conclude that although CLC treatment of dromedary camel sperm improves sperm motility it fails to confer an advantage to them in terms of improved in vitro sperm-oocyte interaction or in vivo fertilization under the conditions tested. Copyright © 2016 Elsevier B.V. All rights reserved.

From laboratory to field studies - The assessment of Biscutella laevigata suitability to biological reclamation of areas contaminated with lead and cadmium.

PubMed

Muszyńska, Ewa; Hanus-Fajerska, Ewa; Piwowarczyk, Barbara; Augustynowicz, Joanna; Ciarkowska, Krystyna; Czech, Tomasz

2017-08-01

The aim of the work was to evaluate the usefulness of the in vitro multiplication of Biscutella laevigata calamine ecotype for in situ reclamation of post-flotation wastes polluted with Pb and Cd. The experiment was conducted on three steps: (i) plant shoots' production under in vitro condition, (ii) establishment of the material in greenhouse experiment, and finally (iii) field cultivation directly on the mining-waste heap of Olkusz Ore-Bearing Region, Poland. This region is known to be one of the most chemically-degraded area in central Europe. The laboratory-set in vitro analysis enabled to obtain the high-quality plant shoots, which multiply the most effectively (with growth tolerance index 130-150%) on medium containing 5.0μM CdCl 2 and 0.5mM Pb(NO 3 ) 2 . These plants were used for the next two ex vitro experiments. Several biometric and physiological analysis (i.e. of photosystem II activity F v /F m and PI, photosynthetic pigment contents) were done to indicate plant physiological status during these experiments. The main novelty of the work was to prove that in vitro-multiplied shoots of B. laevigata - the representative of native flora from Olkusz Ore-Bearing Region - can be successfully implemented in situ for the restoration of these degraded area. Moreover, the addition of sewage sludge as a source of organic compounds significantly improved plants' growth and development what is especially important due to the lack of other legal solutions for the management of the sewage sludge in some countries. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen †

PubMed Central

Crawford, Don L.; Lynch, James M.; Whipps, John M.; Ousley, Margaret A.

1993-01-01

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum. PMID:16349093

Isolation and characterization of actinomycete antagonists of a fungal root pathogen.

PubMed

Crawford, D L; Lynch, J M; Whipps, J M; Ousley, M A

1993-11-01

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum.

Bisphenol A release from orthodontic adhesives measured in vitro and in vivo with gas chromatography.

PubMed

Moreira, Marília Rodrigues; Matos, Leonardo Gontijo; de Souza, Israel Donizeti; Brigante, Tamires Amabile Valim; Queiroz, Maria Eugênia Costa; Romano, Fábio Lourenço; Nelson-Filho, Paulo; Matsumoto, Mírian Aiko Nakane

2017-03-01

The objectives of this study were to quantify in vitro the Bisphenol A (BPA) release from 5 orthodontic composites and to assess in vivo the BPA level in patients' saliva and urine after bracket bonding with an orthodontic adhesive system. For the in-vitro portion of this study, 5 orthodontic composites were evaluated: Eagle Spectrum (American Orthodontics, Sheboygan, Wis), Enlight (Ormco, Orange, Calif), Light Bond (Reliance Orthodontic Products, Itasca, Ill), Mono Lok II (Rocky Mountain Orthodontics, Denver, Colo), and Transbond XT (3M Unitek, Monrovia, Calif). Simulating intraoral conditions, the specimens were immersed in a water/ethanol solution, and the BPA (ng.g -1 ) liberation was measured after 30 minutes, 24 hours, 1 day, 1 week, and 1 month by the gas chromatography system coupled with mass spectrometry. Twenty patients indicated for fixed orthodontic treatment participated in the in-vivo study. Saliva samples were collected before bracket bonding and then 30 minutes, 24 hours, 1 day, 1 week, and 1 month after bonding the brackets. Urine samples were collected before bonding and then at 1 day, 1 week, and 1 month after bonding. The results were analyzed statistically using analysis of variance and Tukey posttest, with a significance level of 5%. All composites evaluated in vitro released small amounts of BPA. Enlight composite showed the greatest release, at 1 month. Regarding the in-vivo study, the mean BPA level in saliva increased significantly only at 30 minutes after bonding in comparison with measurements recorded before bonding. All orthodontic composites released BPA in vitro. Enlight and Light Bond had, respectively, the highest and lowest BPA releases in vitro. The in-vivo experiment showed that bracket bonding with the Transbond XT orthodontic adhesive system resulted in increased BPA levels in saliva and urine. The levels were significant but still lower than the reference dose for daily ingestion. Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

Preparation & in vitro evaluation of 90Y-DOTA-rituximab

PubMed Central

Kameswaran, Mythili; Pandey, Usha; Dash, Ashutosh; Samuel, Grace; Venkatesh, Meera

2016-01-01

Background & objectives: Radioimmunotherapy is extensively being used for the treatment of non-Hodgkin's lymphoma (NHL). Use of rituximab, a chimeric anti-CD20 antibody directed against the CD20 antigen in combination with suitable beta emitters is expected to result in good treatment response by its cross-fire and bystander effects. The present work involves the conjugation of p-isothiocyanatobenzyl DOTA (p-SCN-Bn-DOTA) to rituximab, its radiolabelling with 90Y and in vitro and in vivo evaluation to determine its potential as a radioimmunotherapeutic agent. Methods: Rituximab was conjugated with p-SCN-Bn-DOTA at 1:1 antibody: DOTA molar ratio. The number of DOTA molecules linked to one molecule of rituximab was determined by radioassay and spectroscopic assay. Radiolabelling of rituximab with 90Y was carried out and its in vitro stability was evaluated. In vitro cell binding studies were carried out in Raji cells expressing CD20 antigen. Biodistribution studies were carried out in normal Swiss mice. Results: Using both radioassay and spectroscopic method, it was determined that about five molecules of DOTA were linked to rituximab. Radiolabelling of the rituximab conjugate with 90Y and subsequent purification on PD-10 column gave a product with radiochemical purity (RCP) > 98 per cent which was retained at > 90 per cent up to 72 h when stored at 37°C. In vitro cell binding experiments of 90Y-DOTA-rituximab with Raji cells exhibited specific binding of 20.7 ± 0.1 per cent with 90Y-DOTA-rituximab which reduced to 15.5 ± 0.2 per cent when incubated with cold rituximab. The equilibrium constant Kd for 90Y-DOTA-Rituximab was determined to be 3.38 nM. Radiolabelled antibody showed clearance via hepatobiliary and renal routes and activity in tibia was found to be quite low indicating in vivo stability of 90Y-DOTA-rituximab. Interpretation & conclusions: p-SCN-Bn-DOTA was conjugated with rituximab and radiolabelling with 90Y was carried out. In vitro studies carried out in Raji cells showed the specificity of the radiolabelled conjugate suggesting the potential uitability of the formulation as a radiopharmaceutical for therapy of NHL. PMID:26997015

Preparation & in vitro evaluation of ⁹⁰Y-DOTA-rituximab.

PubMed

Kameswaran, Mythili; Pandey, Usha; Dash, Ashutosh; Samuel, Grace; Venkatesh, Meera

2016-01-01

Radioimmunotherapy is extensively being used for the treatment of non-Hodgkin's lymphoma (NHL). Use of rituximab, a chimeric anti-CD20 antibody directed against the CD20 antigen in combination with suitable beta emitters is expected to result in good treatment response by its cross-fire and bystander effects. The present work involves the conjugation of p-isothiocyanatobenzyl DOTA (p-SCN-Bn-DOTA) to rituximab, its radiolabelling with [90] Y and in vitro and in vivo evaluation to determine its potential as a radioimmunotherapeutic agent. Rituximab was conjugated with p-SCN-Bn-DOTA at 1:1 antibody: DOTA molar ratio. The number of DOTA molecules linked to one molecule of rituximab was determined by radioassay and spectroscopic assay. Radiolabelling of rituximab with 90 Y was carried out and its in vitro stability was evaluated. In vitro cell binding studies were carried out in Raji cells expressing CD20 antigen. Biodistribution studies were carried out in normal Swiss mice. Using both radioassay and spectroscopic method, it was determined that about five molecules of DOTA were linked to rituximab. Radiolabelling of the rituximab conjugate with [90] Y and subsequent purification on PD-10 column gave a product with radiochemical purity (RCP) > 98 per cent which was retained at > 90 per cent up to 72 h when stored at 37°C. In vitro cell binding experiments of 90 Y-DOTA-rituximab with Raji cells exhibited specific binding of 20.7 ± 0.1 per cent with [90] Y-DOTA-rituximab which reduced to 15.5 ± 0.2 per cent when incubated with cold rituximab. The equilibrium constant K d for 90 Y-DOTA-Rituximab was determined to be 3.38 nM. Radiolabelled antibody showed clearance via hepatobiliary and renal routes and activity in tibia was found to be quite low indicating in vivo stability of [90] Y-DOTA-rituximab. p-SCN-Bn-DOTA was conjugated with rituximab and radiolabelling with 90 Y was carried out. In vitro studies carried out in Raji cells showed the specificity of the radiolabelled conjugate suggesting the potential uitability of the formulation as a radiopharmaceutical for therapy of NHL.

Advances on the Transfer of Lipids by Lipid Transfer Proteins.

PubMed

Wong, Louise H; Čopič, Alenka; Levine, Tim P

2017-07-01

Transfer of lipid across the cytoplasm is an essential process for intracellular lipid traffic. Lipid transfer proteins (LTPs) are defined by highly controlled in vitro experiments. The functional relevance of these is supported by evidence for the same reactions inside cells. Major advances in the LTP field have come from structural bioinformatics identifying new LTPs, and from the development of countercurrent models for LTPs. However, the ultimate aim is to unite in vitro and in vivo data, and this is where much progress remains to be made. Even where in vitro and in vivo experiments align, rates of transfer tend not to match. Here we set out some of the advances that might test how LTPs work. Copyright © 2017. Published by Elsevier Ltd.

In Vitro Studies on the Degradability, Bioactivity, and Cell Differentiation of PRP/AZ31B Mg Alloys Composite Scaffold.

PubMed

Zou, Jian; Shi, Zhongmin; Xu, Hongwei; Li, Xiaolin

2017-01-01

In recent years, more and more methods have been developed to improve the bioactivity of the biodegradable materials in bone tissue regeneration. In present study, we used rat mesenchymal stem cells (rMSCs) to evaluate the outcomes of Mg alloys (AZ31B, Magnesium, and Aluminum) and Platelet-rich plasma (PRP)/Mg alloys on rMSCs biocompatibility and osteogenic differentiation. Water absorption experiments indicated that both bare AZ31B and PRP/AZ31B were capable of absorbing large amounts of water. But the water absorption ratio for PRP/AZ31B was significantly higher than that for bare AZ31B. The degradability experiments implied that both samples degraded at same speed. rMSCs on the surface of AZ31B distributed more and better than those on the AZ31B scaffold. In ALP activity experiment, the activity of rMSCs on the PRP/AZ31B was markedly higher than that on the AZ31B scaffolds on the 7th day and 14th day. qRT-PCR also showed that OPN and OCN were expressed in both samples. OPN and OCN expression in PRP/AZ31B sample were higher than those in bare AZ31B samples. In summary, the in vitro study implied that AZ31B combined with PRP could remarkably improve cell seeding, attachment, proliferation, and differentiation.

Thiol functionalized polymethacrylic acid-based hydrogel microparticles for oral insulin delivery.

PubMed

Sajeesh, S; Vauthier, C; Gueutin, C; Ponchel, G; Sharma, Chandra P

2010-08-01

In the present study thiol functionalized polymethacrylic acid-polyethylene glycol-chitosan (PCP)-based hydrogel microparticles were utilized to develop an oral insulin delivery system. Thiol modification was achieved by grafting cysteine to the activated surface carboxyl groups of PCP hydrogels (Cys-PCP). Swelling and insulin loading/release experiments were conducted on these particles. The ability of these particles to inhibit protease enzymes was evaluated under in vitro experimental conditions. Insulin transport experiments were performed on Caco-2 cell monolayers and excised intestinal tissue with an Ussing chamber set-up. Finally, the efficacy of insulin-loaded particles in reducing the blood glucose level in streptozotocin-induced diabetic rats was investigated. Thiolated hydrogel microparticles showed less swelling and had a lower insulin encapsulation efficiency as compared with unmodified PCP particles. PCP and Cys-PCP microparticles were able to inhibit protease enzymes under in vitro conditions. Thiolation was an effective strategy to improve insulin absorption across Caco-2 cell monolayers, however, the effect was reduced in the experiments using excised rat intestinal tissue. Nevertheless, functionalized microparticles were more effective in eliciting a pharmacological response in diabetic animal, as compared with unmodified PCP microparticles. From these studies thiolation of hydrogel microparticles seems to be a promising approach to improve oral delivery of proteins/peptides. Copyright 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Post-Inhibitory Rebound Spikes in Rat Medial Entorhinal Layer II/III Principal Cells: In Vivo, In Vitro, and Computational Modeling Characterization

PubMed Central

Ferrante, Michele; Shay, Christopher F.; Tsuno, Yusuke; William Chapman, G.; Hasselmo, Michael E.

2017-01-01

Abstract Medial entorhinal cortex Layer-II stellate cells (mEC-LII-SCs) primarily interact via inhibitory interneurons. This suggests the presence of alternative mechanisms other than excitatory synaptic inputs for triggering action potentials (APs) in stellate cells during spatial navigation. Our intracellular recordings show that the hyperpolarization-activated cation current (Ih) allows post-inhibitory-rebound spikes (PIRS) in mEC-LII-SCs. In vivo, strong inhibitory-post-synaptic potentials immediately preceded most APs shortening their delay and enhancing excitability. In vitro experiments showed that inhibition initiated spikes more effectively than excitation and that more dorsal mEC-LII-SCs produced faster and more synchronous spikes. In contrast, PIRS in Layer-II/III pyramidal cells were harder to evoke, voltage-independent, and slower in dorsal mEC. In computational simulations, mEC-LII-SCs morphology and Ih homeostatically regulated the dorso-ventral differences in PIRS timing and most dendrites generated PIRS with a narrow range of stimulus amplitudes. These results suggest inhibitory inputs could mediate the emergence of grid cell firing in a neuronal network. PMID:26965902

Hake fish bone as a calcium source for efficient bone mineralization.

PubMed

Flammini, Lisa; Martuzzi, Francesca; Vivo, Valentina; Ghirri, Alessia; Salomi, Enrico; Bignetti, Enrico; Barocelli, Elisabetta

2016-01-01

Calcium is recognized as an essential nutritional factor for bone health. An adequate intake is important to achieve or maintain optimal bone mass in particular during growth and old age. The aim of the present study was to evaluate the efficiency of hake fish bone (HBF) as a calcium source for bone mineralization: in vitro on osteosarcoma SaOS-2 cells, cultured in Ca-free osteogenic medium (OM) and in vivo on young growing rats fed a low-calcium diet. Lithotame (L), a Ca supplement derived from Lithothamnium calcareum, was used as control. In vitro experiments showed that HBF supplementation provided bone mineralization similar to standard OM, whereas L supplementation showed lower activity. In vivo low-Ca HBF-added and L-added diet similarly affected bone deposition. Physico-chemical parameters concerning bone mineralization, such as femur breaking force, tibia density and calcium/phosphorus mineral content, had beneficial effects from both Ca supplementations, in the absence of any evident adverse effect. We conclude HBF derived from by-product from the fish industry is a good calcium supplier with comparable efficacy to L.

Investigation of brain-derived neurotrophic factor (BDNF) gene expression in hypothalamus of obese rats: Modulation by omega-3 fatty acids.

PubMed

Abdel-Maksoud, Sahar M; Hassanein, Sally I; Gohar, Neveen A; Attia, Saad M M; Gad, Mohamed Z

2017-10-01

The aim of this study was investigating the effect of omega-3 fatty acids (ω-3 FAs) on brain-derived neurotrophic factor (BDNF) gene expression, using in vivo and in vitro models, to unravel the potential mechanisms of polyunsaturated fatty acids use in obesity. Twenty-nine Sprague-Dawley rats were divided into three groups; lean controls fed normal chow diet for 14 weeks, obese controls fed 60% of their diet as saturated fats for 14 weeks, and ω-3 FAs-treated rats fed 60% saturated fat diet for 14 weeks with concomitant oral administration of 400 mg/kg/day ω-3 FAs, mainly docosahexaenoic acid and EPA, from week 12 to week 14. For the in vitro experiment, hypothalamic cells from six obese rats were cultured in the presence of different concentrations of ω-3 FAs to determine its direct effect on BDNF expression. In vivo results showed that obesity has negative effect on BDNF gene expression in rat hypothalamus that was reversed by administration of ω-3 FAs. Obese rats showed hypercholesterolemia, hypertriglyceridemia, normoinsulinemia, hyperglycemia and hyperleptinemia. Treatment with ω-3 FAs showed significant decrease in serum total cholesterol and TAG. Also serum glucose level and HOMA index were decreased significantly. In vitro results demonstrated the increase in BDNF expression by ω-3 FAs in a dose-dependent manner. Obesity causes down-regulation of BDNF gene expression that can be reversed by ω-3 FAs treatment, making them an interesting treatment approach for obesity and metabolic disease.

Type IV pili interactions promote intercellular association and moderate swarming of Pseudomonas aeruginosa

PubMed Central

Anyan, Morgen E.; Amiri, Aboutaleb; Harvey, Cameron W.; Tierra, Giordano; Morales-Soto, Nydia; Driscoll, Callan M.; Alber, Mark S.; Shrout, Joshua D.

2014-01-01

Pseudomonas aeruginosa is a ubiquitous bacterium that survives in many environments, including as an acute and chronic pathogen in humans. Substantial evidence shows that P. aeruginosa behavior is affected by its motility, and appendages known as flagella and type IV pili (TFP) are known to confer such motility. The role these appendages play when not facilitating motility or attachment, however, is unclear. Here we discern a passive intercellular role of TFP during flagellar-mediated swarming of P. aeruginosa that does not require TFP extension or retraction. We studied swarming at the cellular level using a combination of laboratory experiments and computational simulations to explain the resultant patterns of cells imaged from in vitro swarms. Namely, we used a computational model to simulate swarming and to probe for individual cell behavior that cannot currently be otherwise measured. Our simulations showed that TFP of swarming P. aeruginosa should be distributed all over the cell and that TFP−TFP interactions between cells should be a dominant mechanism that promotes cell−cell interaction, limits lone cell movement, and slows swarm expansion. This predicted physical mechanism involving TFP was confirmed in vitro using pairwise mixtures of strains with and without TFP where cells without TFP separate from cells with TFP. While TFP slow swarm expansion, we show in vitro that TFP help alter collective motion to avoid toxic compounds such as the antibiotic carbenicillin. Thus, TFP physically affect P. aeruginosa swarming by actively promoting cell−cell association and directional collective motion within motile groups to aid their survival. PMID:25468980

Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

PubMed

Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

2016-01-13

The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds.

On the Determination of Magnesium Degradation Rates under Physiological Conditions.

PubMed

Nidadavolu, Eshwara Phani Shubhakar; Feyerabend, Frank; Ebel, Thomas; Willumeit-Römer, Regine; Dahms, Michael

2016-07-28

The current physiological in vitro tests of Mg degradation follow the procedure stated according to the ASTM standard. This standard, although useful in predicting the initial degradation behavior of an alloy, has its limitations in interpreting the same for longer periods of immersion in cell culture media. This is an important consequence as the alloy's degradation is time dependent. Even if two different alloys show similar corrosion rates in a short term experiment, their degradation characteristics might differ with increased immersion times. Furthermore, studies concerning Mg corrosion extrapolate the corrosion rate from a single time point measurement to the order of a year (mm/y), which might not be appropriate because of time dependent degradation behavior. In this work, the above issues are addressed and a new methodology of performing long-term immersion tests in determining the degradation rates of Mg alloys was put forth. For this purpose, cast and extruded Mg-2Ag and powder pressed and sintered Mg-0.3Ca alloy systems were chosen. DMEM Glutamax +10% FBS (Fetal Bovine Serum) +1% Penicillin streptomycin was used as cell culture medium. The advantages of such a method in predicting the degradation rates in vivo deduced from in vitro experiments are discussed.

Iron Bioavailability Studies of the First Generation of Iron-Biofortified Beans Released in Rwanda.

PubMed

Glahn, Raymond; Tako, Elad; Hart, Jonathan; Haas, Jere; Lung'aho, Mercy; Beebe, Steve

2017-07-21

This paper represents a series of in vitro iron (Fe) bioavailability experiments, Fe content analysis and polyphenolic profile of the first generation of Fe biofortified beans ( Phaseolus vulgaris ) selected for human trials in Rwanda and released to farmers of that region. The objective of the present study was to demonstrate how the Caco-2 cell bioassay for Fe bioavailability can be utilized to assess the nutritional quality of Fe in such varieties and how they may interact with diets and meal plans of experimental studies. Furthermore, experiments were also conducted to directly compare this in vitro approach with specific human absorption studies of these Fe biofortified beans. The results show that other foods consumed with beans, such as rice, can negatively affect Fe bioavailability whereas potato may enhance the Fe absorption when consumed with beans. The results also suggest that the extrinsic labelling approach to measuring human Fe absorption can be flawed and thus provide misleading information. Overall, the results provide evidence that the Caco-2 cell bioassay represents an effective approach to evaluate the nutritional quality of Fe-biofortified beans, both separate from and within a targeted diet or meal plan.

Iron Bioavailability Studies of the First Generation of Iron-Biofortified Beans Released in Rwanda

PubMed Central

Glahn, Raymond; Tako, Elad; Hart, Jonathan; Haas, Jere; Beebe, Steve

2017-01-01

This paper represents a series of in vitro iron (Fe) bioavailability experiments, Fe content analysis and polyphenolic profile of the first generation of Fe biofortified beans (Phaseolus vulgaris) selected for human trials in Rwanda and released to farmers of that region. The objective of the present study was to demonstrate how the Caco-2 cell bioassay for Fe bioavailability can be utilized to assess the nutritional quality of Fe in such varieties and how they may interact with diets and meal plans of experimental studies. Furthermore, experiments were also conducted to directly compare this in vitro approach with specific human absorption studies of these Fe biofortified beans. The results show that other foods consumed with beans, such as rice, can negatively affect Fe bioavailability whereas potato may enhance the Fe absorption when consumed with beans. The results also suggest that the extrinsic labelling approach to measuring human Fe absorption can be flawed and thus provide misleading information. Overall, the results provide evidence that the Caco-2 cell bioassay represents an effective approach to evaluate the nutritional quality of Fe-biofortified beans, both separate from and within a targeted diet or meal plan. PMID:28754026

Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated Adaptations.

PubMed

Danielczok, Jens G; Terriac, Emmanuel; Hertz, Laura; Petkova-Kirova, Polina; Lautenschläger, Franziska; Laschke, Matthias W; Kaestner, Lars

2017-01-01

When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca 2+ -signaling by confocal microscopy of Fluo-4 loaded RBCs. We were able to confirm our in vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca 2+ when RBCs were passing through small capillaries in vivo . Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca 2+ -signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca 2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K + , Cl - , and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constriction.

Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated Adaptations

PubMed Central

Danielczok, Jens G.; Terriac, Emmanuel; Hertz, Laura; Petkova-Kirova, Polina; Lautenschläger, Franziska; Laschke, Matthias W.; Kaestner, Lars

2017-01-01

When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca2+-signaling by confocal microscopy of Fluo-4 loaded RBCs. We were able to confirm our in vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca2+ when RBCs were passing through small capillaries in vivo. Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca2+-signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K+, Cl−, and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constriction. PMID:29259557

Peppermint (Mentha piperita) and albendazole against anisakiasis in an animal model.

PubMed

Romero, Ma Carmen; Navarro, Ma Concepción; Martín-Sánchez, Joaquina; Valero, Adela

2014-12-01

Therapy against anisakiasis requires invasive techniques to extract L3 , and an effective drug against this nematode is needed. The aim of this study was to determine the efficacy of peppermint essential oil (EO) and its main components against the parasite in comparison to albendazole, a drug currently prescribed to treat anisakiasis. We conducted in vitro experiments and studied an experimental model simulating the human infection in Wistar rats. We used polymerase chain reaction restriction fragment length polymorphism to identify A. simplex s.s. and A. pegreffii and determine any differences in their pathogenicity and susceptibility to the treatments. The in vitro and in vivo experiments both showed that the larvicidal activity of peppermint EO, menthol, menthone and menthyl acetate is higher than that of albendazole. Large stomach lesions were observed in 46.7% of the albendazole-treated rats, whereas no gastrointestinal lesions were detected in those treated with peppermint EO, menthol, menthyl acetate or menthone. In this animal model, treatment with peppermint EO or its main components was more effective than was treatment with albendazole. Lesions were more frequently produced by A. simplex s.s. larvae than by A. pegreffii larvae. © 2014 John Wiley & Sons Ltd.

Photodynamic therapy of hamster Greene melanoma in vitro and in vivo using bacteriochlorin-a as photosensitizer

NASA Astrophysics Data System (ADS)

Schuitmaker, J. J.; Van Best, Jaap A.; van Delft, J. L.; Jannink, J. E.; Oosterhuis, J. A.; Vrensen, Gijs F.; Ms Wolff-Rouendaal, Didi; Dubbelman, T. M.

1996-01-01

Efficient photodynamic therapy (PDT) of malignant melanoma may be possible with photosensitizers having absorption maxima in the far-red region e.g., above 700 nm. Bacteriochlorin a (BCA), a non toxic derivative of bacteriochlorphyllin a, has a high molecular absorption coefficient (32.000 M-1.cm-1) at 760 nm. At this wavelength tissue penetration of light is almost optimal and melanin absorption is relatively low. In several series of experiments BCA was proven to be a very effective photosensitizer, in vitro and in vivo. It is preferentially retained in experimental hamster Greene melanoma, rhabdomyosarcoma, RIF- and mamma tumors. Its fluorescence can be detected in vivo, thus enabling early tumor detection and it is rapidly cleared from the tissues which promises no, or minor skin photosensitivity. The effects of BCA-PDT were studied in vitro and in vivo using the heavily pigmented Hamster Greene Melanoma (HGM) cell line as a model. In vitro it was found that the uptake of BCA was time, concentration and temperature dependant. Upon illumination (10 Mw/cm2, 756 nm) after incubation with 2.5 (mu) g/ml BCA for 1 h, almost complete cell kill was obtained within seconds. Hamster Greene Melanoma implanted in the anterior eye chamber of rabbits is an accepted in vivo model for ocular melanoma. The effects of BCA-PDT using this model were studied by light- and electron microscopy. Immediately after PDT intracellular spaces were enlarged and blood vessels were clotted with swollen erythrocytes. Electron microscopy showed fused inner and outer membranes and affected cristae mitchondriales of some mitochondria. With time, the severity of tissue and cell damage increased. One day after irradiation tumor growth had stopped; fluorescein angiography showed non perfusion of the tumor. Histopathology showed almost complete tumor necrosis with occasionally viable cells at the tumor periphery. It is concluded that the direct mitochondrial damage and the vascular damage both contribute to BCA-PDT induced tumor necrosis.

Bone morphogenetic protein 7 (BMP-7) influences tendon-bone integration in vitro.

PubMed

Schwarting, Tim; Lechler, Philipp; Struewer, Johannes; Ambrock, Marius; Frangen, Thomas Manfred; Ruchholtz, Steffen; Ziring, Ewgeni; Frink, Michael

2015-01-01

Successful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration. To study the biological effects of BMP-7 on the process of tendon-bone-integration, two independent in vitro models were used. The first model involved the mono- and coculture of bovine tendon specimens and primary bovine osteoblasts with and without BMP-7 exposure. The second model comprised the mono- and coculture of primary bovine osteoblasts and fibroblasts. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), lactate and osteocalcin (OCN) were analyzed by ELISA. Histological analysis and electron microscopy of the tendon specimens were performed. In both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures. This study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts. Our findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface.

Yerba mate enhances probiotic bacteria growth in vitro but as a feed additive does not reduce Salmonella Enteritidis colonization in vivo.

PubMed

Gonzalez-Gil, Francisco; Diaz-Sanchez, Sandra; Pendleton, Sean; Andino, Ana; Zhang, Nan; Yard, Carrie; Crilly, Nate; Harte, Federico; Hanning, Irene

2014-02-01

Yerba mate (Ilex paraguariensis) is a tea known to have beneficial effects on human health and antimicrobial activity against some foodborne pathogens. Thus, the application of yerba mate as a feed additive for broiler chickens to reduce Salmonella colonization was evaluated. The first in vitro evaluation was conducted by suspending Salmonella Enteritidis and lactic acid bacteria (LAB) in yerba mate extract. The in vivo evaluations were conducted using preventative and horizontal transmission experiments. In all experiments, day-of-hatch chicks were treated with one of the following 1) no treatment (control); 2) ground yerba mate in feed; 3) probiotic treatment (Lactobacillus acidophilus and Pediococcus; 9:1 administered once on day of hatch by gavage); or 4) both yerba mate and probiotic treatments. At d 3, all chicks were challenged with Salmonella Enteritidis (preventative experiment) or 5 of 20 chicks (horizontal transmission experiment). At d 10, all birds were euthanized, weighed, and cecal contents enumerated for Salmonella. For the in vitro evaluation, antimicrobial activity was observed against Salmonella and the same treatment enhanced growth of LAB. For in vivo evaluations, none of the yerba mate treatments significantly reduced Salmonella Enteritidis colonization, whereas the probiotic treatment significantly reduced Salmonella colonization in the horizontal transmission experiment. Yerba mate decreased chicken BW and decreased the performance of the probiotic treatment when used in combination. In conclusion, yerba mate had antimicrobial activity against foodborne pathogens and enhanced the growth of LAB in vitro, but in vivo yerba mate did not decrease Salmonella Enteritidis colonization.

In Vivo Imaging of Experimental Melanoma Tumors using the Novel Radiotracer 68Ga-NODAGA-Procainamide (PCA).

PubMed

Kertész, István; Vida, András; Nagy, Gábor; Emri, Miklós; Farkas, Antal; Kis, Adrienn; Angyal, János; Dénes, Noémi; Szabó, Judit P; Kovács, Tünde; Bai, Péter; Trencsényi, György

2017-01-01

The most aggressive form of skin cancer is the malignant melanoma. Because of its high metastatic potential the early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of the disease. Previous studies have already shown that benzamide derivatives, such as procainamide (PCA) specifically bind to melanin pigment. The aim of this study was to synthesize and investigate the melanin specificity of the novel 68 Ga-labeled NODAGA-PCA molecule in vitro and in vivo using PET techniques. Procainamide (PCA) was conjugated with NODAGA chelator and was labeled with Ga-68 ( 68 Ga-NODAGA-PCA). The melanin specificity of 68 Ga-NODAGA-PCA was tested in vitro , ex vivo and in vivo using melanotic B16-F10 and amelanotic Melur melanoma cell lines. By subcutaneous and intravenous injection of melanoma cells tumor-bearing mice were prepared, on which biodistribution studies and small animal PET/CT scans were performed for 68 Ga-NODAGA-PCA and 18 FDG tracers. 68 Ga-NODAGA-PCA was produced with high specific activity (14.9±3.9 GBq/µmol) and with excellent radiochemical purity (98%<), at all cases. In vitro experiments showed that 68 Ga-NODAGA-PCA uptake of B16-F10 cells was significantly ( p ≤0.01) higher than Melur cells. Ex vivo biodistribution and in vivo PET/CT studies using subcutaneous and metastatic tumor models showed significantly ( p ≤0.01) higher 68 Ga-NODAGA-PCA uptake in B16-F10 primary tumors and lung metastases in comparison with amelanotic Melur tumors. In experiments where 18 FDG and 68 Ga-NODAGA-PCA uptake of B16-F10 tumors was compared, we found that the tumor-to-muscle (T/M) and tumor-to-lung (T/L) ratios were significantly ( p ≤0.05 and p ≤0.01) higher using 68 Ga-NODAGA-PCA than the 18 FDG accumulation. Our novel radiotracer 68 Ga-NODAGA-PCA showed specific binding to the melanin producing experimental melanoma tumors. Therefore, 68 Ga-NODAGA-PCA is a suitable diagnostic radiotracer for the detection of melanoma tumors and metastases in vivo .

In Vivo Imaging of Experimental Melanoma Tumors using the Novel Radiotracer 68Ga-NODAGA-Procainamide (PCA)

PubMed Central

Kertész, István; Vida, András; Nagy, Gábor; Emri, Miklós; Farkas, Antal; Kis, Adrienn; Angyal, János; Dénes, Noémi; Szabó, Judit P.; Kovács, Tünde; Bai, Péter; Trencsényi, György

2017-01-01

Purpose: The most aggressive form of skin cancer is the malignant melanoma. Because of its high metastatic potential the early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of the disease. Previous studies have already shown that benzamide derivatives, such as procainamide (PCA) specifically bind to melanin pigment. The aim of this study was to synthesize and investigate the melanin specificity of the novel 68Ga-labeled NODAGA-PCA molecule in vitro and in vivo using PET techniques. Methods: Procainamide (PCA) was conjugated with NODAGA chelator and was labeled with Ga-68 (68Ga-NODAGA-PCA). The melanin specificity of 68Ga-NODAGA-PCA was tested in vitro, ex vivo and in vivo using melanotic B16-F10 and amelanotic Melur melanoma cell lines. By subcutaneous and intravenous injection of melanoma cells tumor-bearing mice were prepared, on which biodistribution studies and small animal PET/CT scans were performed for 68Ga-NODAGA-PCA and 18FDG tracers. Results: 68Ga-NODAGA-PCA was produced with high specific activity (14.9±3.9 GBq/µmol) and with excellent radiochemical purity (98%<), at all cases. In vitro experiments showed that 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than Melur cells. Ex vivo biodistribution and in vivo PET/CT studies using subcutaneous and metastatic tumor models showed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake in B16-F10 primary tumors and lung metastases in comparison with amelanotic Melur tumors. In experiments where 18FDG and 68Ga-NODAGA-PCA uptake of B16-F10 tumors was compared, we found that the tumor-to-muscle (T/M) and tumor-to-lung (T/L) ratios were significantly (p≤0.05 and p≤0.01) higher using 68Ga-NODAGA-PCA than the 18FDG accumulation. Conclusion: Our novel radiotracer 68Ga-NODAGA-PCA showed specific binding to the melanin producing experimental melanoma tumors. Therefore, 68Ga-NODAGA-PCA is a suitable diagnostic radiotracer for the detection of melanoma tumors and metastases in vivo. PMID:28382139

Patho-physiological evaluation of Duranta erecta for the treatment of urolithiasis.

PubMed

Agawane, Sachin B; Gupta, Vidya S; Kulkarni, Mahesh J; Bhattacharya, Asish K; Koratkar, Santosh S; Rao, Vamkudoth Koteswara

2018-04-07

Urolithiasis is the third common disorder of the urinary system affecting 10-15% of the general population. In recent years, search for new antilithiatic drugs from natural sources has assumed greater importance. This study was performed to investigate the anti-urolithiatic activity of methanolic extract of Duranta erecta leaves by in vitro and in vivo analysis. The study was designed to determine presence of phytochemicals in D. erecta, its yield in percentage, antioxidant activity against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and anti-microbial property against few bacteria. In vitro analysis was carried out study anti-urolithiatic property of D. erecta by nucleation assay and synthetic urine assay for inhibition of calcium oxalate and calcium oxalate monohydrate crystals formation. An in vivo experiment was performed on Wistar rats for confirmation of anti-urolithiatic property of D. erecta in animal model. D. erecta has the presence of primary and secondary metabolites like glycoside, saponins, sterols, flavonoids, phenols, tannins, alkaloids, carbohydrates and proteins. Methanolic extract of D. erecta gave a very good yield (60%). D. erecta proved its antioxidant potential by 93.51% inhibition of DPPH radical at a concentration of 1000 μg/mL where ascorbic showed 94.71% of DPPH radical at the same concentration. In vitro tests like nucleation assay and synthetic urine assay showed that D. erecta inhibits formation of calcium oxalate and calcium oxalate monohydrate crystals. It also showed the anti-microbial property by formation of zone of inhibition against few bacteria. An in vivo experiment on Wistar rat animal model confirmed the anti-urolithiatic property of D. erecta L. leaves extract. Based on the results, we reported that D. erecta may treat calcium oxalate crystal deposition in the kidney by preventing hyperoxaluria-induced peroxidative damage to the renal tubular membrane surface (lipid peroxidation). It has anti-microbial potential so it may also inhibit the secondary bacterial infection in kidney. Based on the data, it can be concluded that this herb can be used as a potential anti-urolithiasis agent for kidney stone removal. Copyright © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights reserved.

Development, characterization, and in vivo assessment of mucoadhesive nanoparticles containing fluconazole for the local treatment of oral candidiasis.

PubMed

Rençber, Seda; Karavana, Sinem Yaprak; Yılmaz, Fethiye Ferda; Eraç, Bayri; Nenni, Merve; Özbal, Seda; Pekçetin, Çetin; Gurer-Orhan, Hande; Hoşgör-Limoncu, Mine; Güneri, Pelin; Ertan, Gökhan

2016-01-01

This study aimed to develop a suitable buccal mucoadhesive nanoparticle (NP) formulation containing fluconazole for the local treatment of oral candidiasis. The suitability of the prepared formulations was assessed by means of particle size (PS), polydispersity index, and zeta potential measurements, morphology analysis, mucoadhesion studies, drug entrapment efficiency (EE), in vitro drug release, and stability studies. Based on the optimum NP formulation, ex vivo drug diffusion and in vitro cytotoxicity studies were performed. Besides, evaluation of the antifungal effect of the optimum formulation was evaluated using agar diffusion method, fungicidal activity-related in vitro release study, and time-dependent fungicidal activity. The effect of the optimum NP formulation on the healing of oral candidiasis was investigated in an animal model, which was employed for the first time in this study. The zeta potential, mucoadhesion, and in vitro drug release studies of various NP formulations revealed that chitosan-coated NP formulation containing EUDRAGIT(®) RS 2.5% had superior properties than other formulations. Concerning the stability study of the selected formulation, the formulation was found to be stable for 6 months. During the ex vivo drug diffusion study, no drug was found in receptor phase, and this is an indication of local effect. The in vitro antifungal activity studies showed the in vitro efficacy of the NP against Candida albicans for an extended period. Also, the formulation had no cytotoxic effect at the tested concentration. For the in vivo experiments, infected rabbits were successfully treated with local administration of the optimum NP formulation once a day. This study has shown that the mucoadhesive NP formulation containing fluconazole is a promising candidate with once-a-day application for the local treatment of oral candidiasis.

Effect of Culture Conditions on Viability of Mouse and Rat Embryos Developed in Vitro

PubMed Central

Popova, Elena; Bader, Michael; Krivokharchenko, Alexander

2011-01-01

Currently in vitro culture of mouse preimplantation embryos has become a very important technique to investigate different mechanisms of early embryogenesis. However, there is a big difference in the preimplantation development between mammalian species. Despite close relatedness to mice, in vitro cultivation of rat preimplantation embryos is still delicate and needs further investigation and optimizations. In this study we have compared the in vitro developmental potential of mouse and rat embryos cultured at different culture conditions in parallel experiments. Interestingly, mouse zygotes developed in vitro until blastocyst stage even in inadequate medium without any phosphates and with low osmolarity which was formulated especially for cultivation of rat embryos. Rat parthenotes and zygotes developed in M16 medium formulated for mouse embryos only till 2-cell stage and further development is blocked completely at this stage. Moreover, developmental ability of rat embryos in vitro was significantly lower in comparison with mouse even in special rat mR1ECM medium. Mouse and rat embryos at 2-cell stage obtained in vivo developed until blastocyst stages significantly more efficiently compared to zygotes. Culture of mouse zygotes in glass capillaries resulted in a significantly higher rate of morula and blastocyst development compared with dishes. The Well-of-the-Well system resulted in a significant improvement when compared with dishes for the culture of rat zygotes only until morula stage. Reduced oxygen tension increased the developmental rate of rat but not mouse zygotes until blastocyst stage. This study demonstrates that development of early preimplantation embryos is altered by different culture conditions and show strong differences even between two related species such as mice and rats. Therefore, for understanding the fundamental mechanisms of early mammalian development it is very important to use embryos of various species. PMID:24710194

Initial in vitro and in vivo evaluation of a self-monitoring prosthetic bypass graft.

PubMed

Neville, Richard F; Gupta, Samit K; Kuraguntla, David J

2017-06-01

Prosthetic grafts used for lower extremity revascularization and dialysis access fail because of hyperplastic stenosis and thrombosis. Graft surveillance is advocated to monitor function; however, graft failure can occur between episodic examinations. An innovative sensor with wireless, microchip technology allows automated surveillance with assessment of graft function using a "cloud"-based algorithm. We performed proof-of-concept experiments with in vitro and in vivo models to assess the feasibility such a real-time graft surveillance system. A self-monitoring graft system was evaluated consisting of a prosthetic conduit of expanded polytetrafluoroethylene and a sensor unit, and a microsensor, microelectronics, battery, and remote processor with a monitor. The sensor unit was integrated on the extraluminal surface of expanded polytetrafluoroethylene grafts without compromise to the lumen of the conduit. The grafts were tested in vitro in a pulsatile, recirculating flow system under physiologic flow parameters. The hemodynamic parameters were varied to assess the ability to obtain wireless signal acquisition reflecting real-time flow properties in vitro. Segments of custom tubing with reduced diameters were inserted into the model to mimic stenosis proximal and distal to the grafts. After characterization of the initial data, the self-monitoring grafts were implanted in an ovine carotid model to assess proof of concept in vivo with 30-day follow-up of signal acquisition as well as arteriographic and histologic analysis. In vitro flow data demonstrated the device was able to determine factors related to prosthetic graft function under varied hemodynamic flow conditions. Wireless signal acquisition using Bluetooth technology (Bluetooth SIG, Inc, Kirkland, Wash) allowed remote data analysis reflecting graft flow parameters through changes in microsensor voltage and frequency. Waveform analysis was applied to construct an algorithm using proprietary software and determine a parameter for graft flow characteristics. This algorithm allowed determination of the degree of stenosis and location of stenosis location (proximal or distal) for display on a remote monitor in real time. Subsequent in vivo experiments confirmed the ability of the system to generate signal acquisition through skin and soft tissue under biologic conditions with no arteriographic stenosis and a favorable healing response at 30-day harvest. Initial in vitro and in vivo experiments demonstrate the ability for a self-monitoring graft system to remotely monitor hemodynamic parameters reflecting graft function using wireless data transmission. This automated system shows promise to deliver real-time data that can be analyzed by cloud-based algorithms alerting the clinician of a change in graft function or development of stenosis for further diagnostic study or intervention before graft failure. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

PRK by Er:YAG laser: in-vitro studies and first in-vivo experiences

NASA Astrophysics Data System (ADS)

Steiner, Rudolf W.; Leiacker, Richard; Russ, Detlef; Seiler, Theo

1996-01-01

Photorefractive keratectomy (PRK) is usually performed by an excimer laser at 193 nm wavelength. Ablatio of corneal tissue is, however, not only possible in the UV region of the optical spectrum but also in the IR where water is an excellent absorber. Therefore, an Er:YAG laser was used at 2.94 micrometer wavelength as an alternative laser light source to perform in vitro studies of corneal ablation and also first clinical experiments to correct myopia of patients with blind eyes.

Skylab

NASA Image and Video Library

1973-01-01

This chart describes the Skylab student experiment In-Vitro Immunology, proposed by Todd A. Meister of Jackson Heights, New York. He suggested an in-vitro observation of the effects of zero-gravity on a presipitin-type antigen-antibody reaction, as compared with the same reaction carried out in an Earth-based laboratory. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro.

PubMed

Yin, Jikai; Cai, Zhongliang; Zhang, Li; Zhang, Jian; He, Xianli; Du, Xilin; Wang, Qing; Lu, Jianguo

2013-03-01

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

Independent Epileptiform Discharge Patterns in the Olfactory and Limbic Areas of the In Vitro Isolated Guinea Pig Brain During 4-Aminopyridine Treatment

PubMed Central

Carriero, Giovanni; Uva, Laura; Gnatkovsky, Vadym; Avoli, Massimo; de Curtis, Marco

2016-01-01

In vitro studies performed on brain slices demonstrate that the potassium channel blocker 4-aminopyridine (4AP, 50 μM) discloses electrographic seizure activity and interictal discharges. These epileptiform patterns have been further analyzed here in a isolated whole guinea pig brain in vitro by using field potential recordings in olfactory and limbic structures. In 8 of 13 experiments runs of fast oscillatory activity (fast runs, FRs) in the piriform cortex (PC) propagated to the lateral entorhinal cortex (EC), hippocampus and occasionally to the medial EC. Early and late FRs were asynchronous in the hemispheres showed different duration [1.78 ± 0.51 and 27.95 ± 4.55 (SD) s, respectively], frequency of occurrence (1.82 ± 0.49 and 34.16 ± 6.03 s) and frequency content (20–40 vs. 40–60 Hz). Preictal spikes independent from the FRs appeared in the hippocampus/EC and developed into ictal-like discharges that did not propagate to the PC. Ictal-like activity consisted of fast activity with onset either in the hippocampus (n = 6) or in the mEC (n = 2), followed by irregular spiking and sequences of diffusely synchronous bursts. Perfusion of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid (100 μM) did not prevent FRs, increased the duration of limbic ictal-like discharges and favored their propagation to olfactory structures. The AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (50 μM) blocked ictal-like events and reduced FRs. In conclusion, 4AP-induced epileptiform activities are asynchronous and independent in olfactory and hippocampal-entorhinal regions. Epileptiform discharges in the isolated guinea pig brain show different pharmacological properties compared with rodent in vitro slices. PMID:20220076

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

1988-05-03

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purifiedmore » fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.« less

Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks.

PubMed

Borm, Paul J A; Cakmak, Gonca; Jermann, Erich; Weishaupt, Christel; Kempers, Pascal; van Schooten, Frederik Jan; Oberdörster, Günter; Schins, Roel P F

2005-06-01

The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m(3)). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 microM) and a mixture of 16 PAHs (0.1 microM) were used. No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 microg/cm(2)) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely.

CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer

PubMed Central

Yoshida, Kazumichi; Tsujimoto, Hironori; Matsumura, Kouji; Kinoshita, Manabu; Takahata, Risa; Matsumoto, Yusuke; Hiraki, Shuichi; Ono, Satoshi; Seki, Shuhji; Yamamoto, Junji; Hase, Kazuo

2015-01-01

CD47 is an antiphagocytic molecule that acts via ligation to signal regulatory protein alpha on phagocytes; its enhanced expression and therapeutic targeting have recently been reported for several malignancies. However, CD47 expression in gastric cancer is not well documented. Immunohistochemical expression of CD47 in surgical specimens was investigated. Expression of CD47 and CD44, a known gastric cancer stem cell marker, were investigated in gastric cancer cell lines by flow cytometry. MKN45 and MKN74 gastric cancer cells were sorted by fluorescence-activated cell sorting according to CD44 and CD47 expression levels, and their in vitro proliferation, spheroid-forming capacity, and in vivo tumorigenicity were studied. In vitro phagocytosis of cancer cells by human macrophages in the presence of a CD47 blocking monoclonal antibody (B6H12) and the survival of immunodeficient mice intraperitoneally engrafted with MKN45 cells and B6H12 were compared to experiments using control antibodies. Immunohistochemistry of the clinical specimens indicated that CD47 was positive in 57 out of 115 cases, and its positivity was an independent adverse prognostic factor. Approximately 90% of the MKN45 and MKN74 cells expressed CD47 and CD44. CD47hi gastric cancer cells showed significantly higher proliferation and spheroid colony formation than CD47lo, and CD44hiCD47hi cells showed the highest proliferation in vitro and tumorigenicity in vivo. B6H12 significantly enhanced in vitro phagocytosis of cancer cells by human macrophages and prolonged the survival of intraperitoneal cancer dissemination in mice compared to control antibodies. In conclusion, CD47 is an adverse prognostic factor and promising therapeutic target in gastric cancer. PMID:26077800

Elucidation of Arctigenin Pharmacokinetics and Tissue Distribution after Intravenous, Oral, Hypodermic and Sublingual Administration in Rats and Beagle Dogs: Integration of In Vitro and In Vivo Findings.

PubMed

Li, Jie; Li, Xin; Ren, Yu-Shan; Lv, Yuan-Yuan; Zhang, Jun-Sheng; Xu, Xiao-Li; Wang, Xian-Zhen; Yao, Jing-Chun; Zhang, Gui-Min; Liu, Zhong

2017-01-01

Although arctigenin ( AG ) has diverse bioactivities, such as anti-oxidant, anti-inflammatory, anti-cancer, immunoregulatory and neuroprotective activities, its pharmacokinetics have not been systematically evaluated. The purpose of this work was to identify the pharmacokinetic properties of AG via various experiments in vivo and in vitro . In this research, rats and beagle dogs were used to investigate the PK (pharmacokinetics, PK) profiles of AG with different drug-delivery manners, including intravenous (i.v), hypodermic injection (i.h), and sublingual (s.l) administration. The data shows that AG exhibited a strong absorption capacity in both rats and beagle dogs (absorption rate < 1 h), a high absorption degree (absolute bioavailability > 100%), and a strong elimination ability ( t 1/2 < 2 h). The tissue distributions of AG at different time points after i.h showed that the distribution of AG in rat tissues is rapid (2.5 h to reach the peak) and wide (detectable in almost all tissues and organs). The AG concentration in the intestine was the highest, followed by that in the heart, liver, pancreas, and kidney. In vitro , AG were incubated with human, monkey, beagle dog and rat liver microsomes. The concentrations of AG were detected by UPLC-MS/MS at different time points (from 0 min to 90 min). The percentages of AG remaining in four species' liver microsomes were human (62 ± 6.36%) > beagle dog (25.9 ± 3.24%) > rat (15.7 ± 9%) > monkey (3.69 ± 0.12%). This systematic investigation of pharmacokinetic profiles of arctigenin (AG) in vivo and in vitro is worthy of further exploration.

Elucidation of Arctigenin Pharmacokinetics and Tissue Distribution after Intravenous, Oral, Hypodermic and Sublingual Administration in Rats and Beagle Dogs: Integration of In Vitro and In Vivo Findings

PubMed Central

Li, Jie; Li, Xin; Ren, Yu-Shan; Lv, Yuan-Yuan; Zhang, Jun-Sheng; Xu, Xiao-Li; Wang, Xian-Zhen; Yao, Jing-Chun; Zhang, Gui-Min; Liu, Zhong

2017-01-01

Although arctigenin (AG) has diverse bioactivities, such as anti-oxidant, anti-inflammatory, anti-cancer, immunoregulatory and neuroprotective activities, its pharmacokinetics have not been systematically evaluated. The purpose of this work was to identify the pharmacokinetic properties of AG via various experiments in vivo and in vitro. In this research, rats and beagle dogs were used to investigate the PK (pharmacokinetics, PK) profiles of AG with different drug-delivery manners, including intravenous (i.v), hypodermic injection (i.h), and sublingual (s.l) administration. The data shows that AG exhibited a strong absorption capacity in both rats and beagle dogs (absorption rate < 1 h), a high absorption degree (absolute bioavailability > 100%), and a strong elimination ability (t1/2 < 2 h). The tissue distributions of AG at different time points after i.h showed that the distribution of AG in rat tissues is rapid (2.5 h to reach the peak) and wide (detectable in almost all tissues and organs). The AG concentration in the intestine was the highest, followed by that in the heart, liver, pancreas, and kidney. In vitro, AG were incubated with human, monkey, beagle dog and rat liver microsomes. The concentrations of AG were detected by UPLC-MS/MS at different time points (from 0 min to 90 min). The percentages of AG remaining in four species’ liver microsomes were human (62 ± 6.36%) > beagle dog (25.9 ± 3.24%) > rat (15.7 ± 9%) > monkey (3.69 ± 0.12%). This systematic investigation of pharmacokinetic profiles of arctigenin (AG) in vivo and in vitro is worthy of further exploration. PMID:28659807

IN VITRO KILLING OF PERKINSUS MARINUS BY HEMOCYTES OF OYSTERS CRASSOSTREA VIRGINICA

EPA Science Inventory

A colorimetric microbicidal assay was adapted, optimized and applied in experiments to characterize the in vitro capacity of eastern oyster (Crassostrea virginica) hemocytes to kill cultured isolates of Perkinsus marinus, a protozoan parasite causing a highly destructive disease...

Targeting Src and tubulin in mucinous ovarian carcinoma

PubMed Central

Liu, Tao; Hu, Wei; Dalton, Heather J.; Choi, Hyun Jin; Huang, Jie; Kang, Yu; Pradeep, Sunila; Miyake, Takahito; Song, Jian H.; Wen, Yunfei; Lu, Chunhua; Pecot, Chad V.; Bottsford-Miller, Justin; Zand, Behrouz; Jennings, Nicholas B; Ivan, Cristina; Gallick, Gary E.; Baggerly, Keith A; Hangauer, David G.; Coleman, Robert L.; Frumovitz, Michael; Sood, Anil K.

2013-01-01

Purpose To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma. Experimental design The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models. Results In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2/M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01-sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules. Conclusion Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma. PMID:24100628

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

PubMed

Duan, Dongyu; Wang, Aiping; Ni, Ling; Zhang, Liping; Yan, Xiuju; Jiang, Ying; Mu, Hongjie; Wu, Zimei; Sun, Kaoxiang; Li, Youxin

2018-01-01

Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab' (antigen-binding fragments cut from TMAB)-modified NPs (Fab'-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab'-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The release kinetics showed that both Fab'-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab'-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab'-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2-) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab'-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab'-Cur-NPs was higher than that of TMAB-Cur-NPs. Fab' fragment has greater capacity than the intact antibody to achieve tumor targeting through NP-based delivery.

Chitinolytic Streptomyces vinaceusdrappus S5MW2 isolated from Chilika lake, India enhances plant growth and biocontrol efficacy through chitin supplementation against Rhizoctonia solani.

PubMed

Yandigeri, Mahesh S; Malviya, Nityanand; Solanki, Manoj Kumar; Shrivastava, Pooja; Sivakumar, G

2015-08-01

A chitinolytic actinomycete Streptomyces vinaceusdrappus S5MW2 was isolated from water sample of Chilika lake, India and identified using 16S rRNA gene sequencing. It showed in vitro antifungal activity against the sclerotia producing pathogen Rhizoctonia solani in a dual culture assay and by chitinase enzyme production in a chitin supplemented minimal broth. Moreover, isolate S5MW2 was further characterized for biocontrol (BC) and plant growth promoting features in a greenhouse experiment with or without colloidal chitin (CC). Results of greenhouse experiment showed that CC supplementation with S5MW2 showed a significant growth of tomato plants and superior disease reduction as compared to untreated control and without CC treated plants. Moreover, higher accumulation of chitinase also recovered in the CC supplemented plants. Significant effect of CC also concurred with the Analysis of Variance of greenhouse parameters. These results show that the a marine antagonist S5MW2 has BC efficiency against R. solani and chitinase enzyme played important role in plant resistance.

Influence of a new surface modification of intraocular lenses with fluoroalkylsilan on the adherence of endophthalmitis-causing bacteria in vitro.

PubMed

Kienast, Antonia; Kämmerer, Regine; Weiss, Claudia; Klinger, Matthias; Menz, Dirk-Henning; Dresp, Joachim; Ohgke, Helge; Solbach, Werner; Laqua, Horst; Hoerauf, Hans

2006-09-01

Dynasilan is a fluoroalkylsilan that is able to interact with surface active centres on intraocular lenses (IOL), offering a new way for surface modification of different IOL materials. The purpose of this in vitro study was to investigate the influence of this new surface modification on the adherence of two typical endophthalmitis causing bacteria (Staphylococcus epidermidis, Propionibacterium acnes). In a pilot experiment, the effect of Dynasilan coating on the adherence of S. epidermidis was tested on glass slides. Forty-two Dynasilan-modified and 42 unmodified IOL (14 PMMA, 14 silicone and 14 hydrogel) were incubated at 37 degrees C in brain heart infusion broth (10(8) CFU/ml) with either S. epidermidis for 24 h or with P. acnes for 1 h. Subsequently, the adherent bacteria were resuspended using ultrasonification at 35 kHz for 3x45 s. After dilution series and incubation at 37 degrees C on Petri dishes for 24 h and 3 days, respectively, the colonies were counted. In the pilot experiment, a markedly lower number of adherent S. epidermidis was observed on Dynasilan-modified glass slides. Of all IOL materials incubated with S. epidermidis, those modified with Dynasilan showed a lower mean number of adherent bacteria (mean 1.37x10(7); SD 2.37x10(7)) than those untreated (2.43x10(7); SD 3.04x10(7)). IOLs incubated with P. acnes showed a significantly lower mean number of adherent bacteria of 2.51x10(4) (SD 2.71x10(4)) on Dynasilan-modified IOLs versus 6.27x10(4) (SD 7.70x10(4)) on untreated IOLs. The presented in vitro results indicate that Dynasilan surface modification is able to reduce the adherence of S. epidermidis and P. acnes on all IOL materials tested. Further studies regarding the stability of this modification and its biocompatibility must be performed.

Inhibitory effects of trehalose on fibroblast proliferation and implications for ocular surgery.

PubMed

Takeuchi, Kimio; Nakazawa, Mitsuru; Ebina, Yuichi; Sato, Kota; Metoki, Tomomi; Miyagawa, Yasuhiro; Ito, Tadashi

2010-11-01

Trehalose is a disaccharide which plays an important role in preserving cells from completely dehydrated circumstances. In this study, we investigated effects of trehalose on proliferative activity of fibroblasts and epithelial cells both in vitro and in vivo. As in vitro assessment, normal human dermal fibroblasts and normal human epidermal keratinocytes were cultured in media containing various concentrations of trehalose. Growth activities of cells were evaluated with MTT assay and diff-quick™ staining. Expressions of vimentin and α smooth muscle actin (α-SMA) changed by trehalose were semiquantitatively measured by Western blot. As an in vivo study, 5% or 10% trehalose was topically instilled onto rabbit eyes after simple conjunctival incision or trabeculectomy. Condition of the surgical wound was evaluated by morphologically and immunohistochemically using isolectin B4 and antibodies specific for vimentin and α-SMA. Intraocular pressures (IOPs) after trabeculectomy were compared between eyes treated with trehalose and 0.04% mitomycin C (MMC). Results obtained by in vitro experiments showed that growth activities of cultured fibroblasts and keratinocytes were inhibited by trehalose in a dose-dependent manner. Fibroblasts were strongly inhibited by trehalose concentrations ≧ 5% of trehalose, whereas keratinocytes were less inhibited compared to fibroblasts. Expressions of vimentin and α-SMA were reduced by trehalose. With in vivo experiments, postoperative application of trehalose resulted in less firm adhesion between conjunctiva and sclera compared to controls. Immunohistochemical studies showed reduced staining of isolectin B4, vimentin and α-SMA in conjunctival wounds treated by topical trehalose. Also, after trabeculectomy, IOP remained in a low range during instillation of topical trehalose solution. We concluded that trehalose has inhibitory effects on proliferation of fibroblasts and vascular tissues, partially due to inhibition of transformation of fibroblasts into myofibroblasts in wound tissues. The present results imply that trehalose can be a potential agent for preventing postoperative fibrous scar formation after ocular surgery such as glaucoma filtration surgery. Copyright © 2010 Elsevier Ltd. All rights reserved.

Bioactive and thermally compatible glass coating on zirconia dental implants.

PubMed

Kirsten, A; Hausmann, A; Weber, M; Fischer, J; Fischer, H

2015-02-01

The healing time of zirconia implants may be reduced by the use of bioactive glass coatings. Unfortunately, existing glasses are either bioactive like Bioglass 45S5 but thermally incompatible with the zirconia substrate, or they are thermally compatible but exhibit only a very low level of bioactivity. In this study, we hypothesized that a tailored substitution of alkaline earth metals and alkaline metals in 45S5 can lead to a glass composition that is both bioactive and thermally compatible with zirconia implants. A novel glass composition was analyzed using x-ray fluorescence spectroscopy, dilatometry, differential scanning calorimetry, and heating microscopy to investigate its chemical, physical, and thermal properties. Bioactivity was tested in vitro using simulated body fluid (SBF). Smooth and microstructured glass coatings were applied using a tailored spray technique with subsequent thermal treatment. Coating adhesion was tested on implants that were inserted in bovine ribs. The cytocompatibility of the coating was analyzed using L929 mouse fibroblasts. The coefficient of thermal expansion of the novel glass was shown to be slightly lower (11.58 · 10(-6) K(-1)) than that of the zirconia (11.67 · 10(-6) K(-1)). After storage in SBF, the glass showed reaction layers almost identical to the bioactive glass gold standard, 45S5. A process window between 800 °C and 910 °C was found to result in densely sintered and amorphous coatings. Microstructured glass coatings on zirconia implants survived a minimum insertion torque of 60 Ncm in the in vitro experiment on bovine ribs. Proliferation and cytotoxicity of the glass coatings was comparable with the controls. The novel glass composition showed a strong adhesion to the zirconia substrate and a significant bioactive behavior in the SBF in vitro experiments. Therefore, it holds great potential to significantly reduce the healing time of zirconia dental implants. © International & American Associations for Dental Research 2014.

Human induced pluripotent stem cell‐derived versus adult cardiomyocytes: an in silico electrophysiological study on effects of ionic current block

PubMed Central

Paci, M; Hyttinen, J; Rodriguez, B

2015-01-01

Background and Purpose Two new technologies are likely to revolutionize cardiac safety and drug development: in vitro experiments on human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) and in silico human adult ventricular cardiomyocyte (hAdultV‐CM) models. Their combination was recently proposed as a potential replacement for the present hERG‐based QT study for pharmacological safety assessments. Here, we systematically compared in silico the effects of selective ionic current block on hiPSC‐CM and hAdultV‐CM action potentials (APs), to identify similarities/differences and to illustrate the potential of computational models as supportive tools for evaluating new in vitro technologies. Experimental Approach In silico AP models of ventricular‐like and atrial‐like hiPSC‐CMs and hAdultV‐CM were used to simulate the main effects of four degrees of block of the main cardiac transmembrane currents. Key Results Qualitatively, hiPSC‐CM and hAdultV‐CM APs showed similar responses to current block, consistent with results from experiments. However, quantitatively, hiPSC‐CMs were more sensitive to block of (i) L‐type Ca2+ currents due to the overexpression of the Na+/Ca2+ exchanger (leading to shorter APs) and (ii) the inward rectifier K+ current due to reduced repolarization reserve (inducing diastolic potential depolarization and repolarization failure). Conclusions and Implications In silico hiPSC‐CMs and hAdultV‐CMs exhibit a similar response to selective current blocks. However, overall hiPSC‐CMs show greater sensitivity to block, which may facilitate in vitro identification of drug‐induced effects. Extrapolation of drug effects from hiPSC‐CM to hAdultV‐CM and pro‐arrhythmic risk assessment can be facilitated by in silico predictions using biophysically‐based computational models. PMID:26276951

Neoplastic transformation of SV40-immortalized human urinary tract epithelial cells by in vitro exposure to 3-methylcholanthrene.

PubMed

Reznikoff, C A; Loretz, L J; Christian, B J; Wu, S Q; Meisner, L F

1988-08-01

Normal human urinary tract epithelial cells (HUC) were neoplastically transformed in vitro using a step-wise strategy. First, a partially transformed non-virus-producing cell line was obtained after infection of HUC with simian virus 40 (SV40). This cell line (SV-HUC-1) was demonstrated to be clonal in origin, as 100% of cells contained at least five of seven marker chromosomes. Marker chromosomes were formed by balanced translocations resulting in a 'pseudodiploid' cell line. SV-HUC-1 showed altered growth properties in vitro (e.g. anchorage independent growth) but failed to form tumors in athymic nude mice, even after 3 years in culture (80 passages). In the studies reported here, SV-HUC-1 at early passages (P15-P19) were exposed to 3-methylcholanthrene (MCA) in three separate experiments. After a six-week post-treatment period of cell culture, cells were inoculated s.c. into athymic nude mice. In all experiments, MCA-treated SV-HUC-1 formed carcinomas in mice usually with a latent period of 5-8 weeks. These carcinomas showed heterogeneity with respect to histopathologies and growth properties in the mice and karyotypes. All the tumors retained SV-HUC-1 chromosome markers, but each independent transformant was aneuploid and contained unique new marker chromosomes. Chromosomes usually altered in tumor cells included numbers 3, 5, 6, 9, 11 and 13. Mutations in the ras family of cellular proto-oncogenes resulting in altered mobility of the p21 protein product were not detected in six cell lines established from independently derived tumors. It is not yet known whether other cellular proto-oncogenes are activated in these tumorigenic transformants. Neither control SV-HUC-1 (which were not exposed to MCA), nor early passage HUC exposed to MCA formed tumors when inoculated into mice. Thus, the tumorigenic transformation of HUC resulted from the combined actions of SV40 and MCA.

Bioactive and Thermally Compatible Glass Coating on Zirconia Dental Implants

PubMed Central

Kirsten, A.; Hausmann, A.; Weber, M.; Fischer, J.

2015-01-01

The healing time of zirconia implants may be reduced by the use of bioactive glass coatings. Unfortunately, existing glasses are either bioactive like Bioglass 45S5 but thermally incompatible with the zirconia substrate, or they are thermally compatible but exhibit only a very low level of bioactivity. In this study, we hypothesized that a tailored substitution of alkaline earth metals and alkaline metals in 45S5 can lead to a glass composition that is both bioactive and thermally compatible with zirconia implants. A novel glass composition was analyzed using x-ray fluorescence spectroscopy, dilatometry, differential scanning calorimetry, and heating microscopy to investigate its chemical, physical, and thermal properties. Bioactivity was tested in vitro using simulated body fluid (SBF). Smooth and microstructured glass coatings were applied using a tailored spray technique with subsequent thermal treatment. Coating adhesion was tested on implants that were inserted in bovine ribs. The cytocompatibility of the coating was analyzed using L929 mouse fibroblasts. The coefficient of thermal expansion of the novel glass was shown to be slightly lower (11.58·10–6 K–1) than that of the zirconia (11.67·10–6 K–1). After storage in SBF, the glass showed reaction layers almost identical to the bioactive glass gold standard, 45S5. A process window between 800 °C and 910 °C was found to result in densely sintered and amorphous coatings. Microstructured glass coatings on zirconia implants survived a minimum insertion torque of 60 Ncm in the in vitro experiment on bovine ribs. Proliferation and cytotoxicity of the glass coatings was comparable with the controls. The novel glass composition showed a strong adhesion to the zirconia substrate and a significant bioactive behavior in the SBF in vitro experiments. Therefore, it holds great potential to significantly reduce the healing time of zirconia dental implants. PMID:25421839

Elevated CO(2) and drought stress effects on the chemical composition of maize plants, their ruminal fermentation and microbial diversity in vitro.

PubMed

Meibaum, Birgit; Riede, Susanne; Schröder, Bernd; Manderscheid, Remy; Weigel, Hans-Joachim; Breves, Gerhard

2012-12-01

Climate changes are supposed to influence productivity and chemical composition of plants. In the present experiments, it was hypothesised that the incubation of plants exposed to elevated atmospheric carbon dioxide concentrations ([CO₂]) and drought stress will result in different ruminal fermentation pattern and microbial diversity compared to unaffected plants. Maize plants were grown, well-watered under ambient (380 ppm CO₂, Variant A) and elevated [CO₂] (550 ppm CO₂, Variant B). Furthermore, each CO₂ treatment was also exposed to drought stress (380 ppm and 550 ppm CO₂,Variants C and D, respectively), which received only half as much water as the well-watered plants. Plant material from these treatments was incubated in a semi-continuous in vitro fermentation experiment using the rumen simulation technique. Single strand conformation polymorphism (SSCP) analysis was conducted for Bacteria and Archaea specific profiles. The analysis of crude nutrients showed higher contents of fibre fraction in drought stress Variants C and D. Crude protein content was increased by drought stress under ambient but not under elevated [CO₂]. Fermentation of drought stress variants resulted in significantly increased pH values, decreased digestibilities of organic matter and increased ammonia-N (NH₃-N) concentrations compared with well-watered variants. Additionally, the 550 ppm CO₂ Variants B and D showed significantly lower NH₃-N concentrations than Variants A and C. The Bacteria- and Archaea-specific SSCP profiles as well as the production rates of short-chain fatty acids and their molar percentages were not affected by treatments. During the first four days of equilibration period, a decrease of molar percentage of acetate and increased molar percentages of propionate were observed for all treatments. These alterations might have been induced by adaptation of the in vitro system to the new substrate. The rumen microflora appeared to be highly adaptive and could cope with altered contents of crude nutrients in plants as induced by elevated [CO₂] and drought stress.

Biochemical Characterization of Fungus Isolated during In vitro Propagation of Bambusa balcooa.

PubMed

Tyagi, Bhawna; Tewari, Salil; Dubey, Ashutosh

2018-01-01

Bambusa balcooa ( Poaceae: Bambusoideae ) is a multipurpose bamboo species, which is native of the Indian subcontinent. B. balcooa is regarded as one of the best species for scaffolding and building purposes because of its strong culm. Other uses include paper pulp, handicrafts, and products of the wood chip industry. Due to these various uses in industries, this species has been identified as one of the priority bamboos by the National Bamboo Mission. This study is designed to analyze the identification of fungus and develop the strategy to eliminate the contamination during in vitro establishment of B. balcooa through nodal part. Fungus contamination is a problem which is encountered during in vitro establishment of B. balcooa cultures. In the present study, fungus contamination from in vitro cultured plant has been isolated and subjected to partial sequence analysis of the 18S rRNA gene to identify the fungus strain. Experiments were designed to develop a strategy for removal of the fungus contamination with the help of antifungal compounds and commercial antimicrobial supplement supplied by HiMedia. Fusarium equiseti was identified as endophytic fungus. It was observed that antimicrobial supplement at concentration of 500 μl/l was more effective concentration to remove fungus contamination and not showed any detrimental effect on growth parameters of shoot. This experiment would help in identification and to get rid of fungal contamination and improve the in vitro establishment of B. balcooa cultures for large-scale propagation. Endogenous fungus was isolated from contaminated culture of B. balcooa , and it was identified as Fusarium equiseti and submitted to NCBI under accession no. KP274872. The endophytic fungus had shown substantial production of amylase, cellulase, and protease media. Gibberellic acid (GA 3 ) production by F. equiseti was maximum on the 7 th day on inoculation. Abbreviations used: B. balcooa : Bambusa balcooa , F. equiseti : Fusarium equiseti , PDA: Potato dextrose agar, PCR: Polymerase chain reaction, MS: Murashige and Skoog's, BAP: 6-Benzylaminopurine, ITS1/4: Internal transcribed spacer region 1/4, GA 3 : Gibberellic acid.

Utility of a tumor-mimic model for the evaluation of the accuracy of HIFU treatments. results of in vitro experiments in the liver.

PubMed

N'Djin, William Apoutou; Melodelima, David; Parmentier, Hubert; Chesnais, Sabrina; Rivoire, Michel; Chapelon, Jean Yves

2008-12-01

Presented in this article is a tumor-mimic model that allows the evaluation, before clinical trials, of the targeting accuracy of a high intensity focused ultrasound (HIFU) device for the treatment of the liver. The tumor-mimic models are made by injecting a warm solution that polymerizes in hepatic tissue and forms a 1 cm discrete lesion that is detectable by ultrasound imaging and gross pathology. First, the acoustical characteristics of the tumor-mimics model were measured in order to determine if this model could be used as a target for the evaluation of the accuracy of HIFU treatments without modifying HIFU lesions in terms of size, shape and homogeneity. On average (n = 10), the attenuation was 0.39 +/- 0.05 dB.cm(-1) at 1 MHz, the ultrasound propagation velocity was 1523 +/- 1 m.s(-1) and the acoustic impedance was 1.84 +/- 0.00 MRayls. Next, the tumor-mimic models were used in vitro in order to verify, at a preclinical stage, that lesions created by HIFU devices guided by ultrasound imaging are properly positioned in tissues. The HIFU device used in this study is a 256-element phased-array toroid transducer working at a frequency of 3 MHz with an integrated ultrasound imaging probe working at a frequency of 7.5 MHz. An initial series of in vitro experiments has shown that there is no significant difference in the dimensions of the HIFU lesions created in the liver with or without tumor-mimic models (p = 0.3049 and p = 0.8796 for the diameter and depth, respectively). A second in vitro study showed that HIFU treatments performed on five tumor-mimics with safety margins of at least 1 mm were properly positioned. The margins obtained were on average 9.3 +/- 2.7 mm (min. 3.0 - max. 20.0 mm). This article presents in vitro evidence that these tumor-mimics are identifiable by ultrasound imaging, they do not modify the geometry of HIFU lesions and, thus, they constitute a viable model of tumor-mimics indicated for HIFU therapy.

Influence of Starting Powders on Hydroxyapatite Coatings Fabricated by Room Temperature Spraying Method.

PubMed

Seo, Dong Seok; Lee, Jong Kook; Hwang, Kyu Hong; Hahn, Byung Dong; Yoon, Seog Young

2015-08-01

Three types of raw materials were used for the fabrication of hydroxyapatite coatings by using the room temperature spraying method and their influence on the microstructure and in vitro characteristics were investigated. Starting hydroxyapatite powders for coatings on titanium substrate were prepared by a heat treatment at 1100 °C for 2 h of bovine bone, bone ash, and commercial hydroxyapatite powders. The phase compositions and Ca/P ratios of the three hydroxyapatite coatings were similar to those of the raw materials without decomposition or formation of a new phase. All hydroxyapatite coatings showed a honeycomb structure, but their surface microstructures revealed different features in regards to surface morphology and roughness, based on the staring materials. All coatings consisted of nano-sized grains and had dense microstructure. Inferred from in vitro experiments in pure water, all coatings have a good dissolution-resistance and biostability in water.

Control of rabbit dura mater optical properties with osmotical liquids

NASA Astrophysics Data System (ADS)

Yao, Lei; Cheng, Haiying; Luo, Qingming; Zhang, Wei; Zeng, Shaoqun; Tuchin, Valery V.

2002-04-01

An experimental study of controlling the optical properties of in vitro and in vivo rabbit dura mater with administration of osmotical agents, 40% glucose solution and glycerol, using video camera and spectrometer was presented. The preliminary results of experimental study of influence of osmotical liquids (glucose solutions, glycerol) on transmittance (in vitro) and reflectance (in vivo) spectra of rabbit dura mater were reported. The significant decreasing of the reflectance and increasing of the transmittance of dura mater under action of osmotical solutions were demonstrated. Experiments showed that administration of osmolytes to dura mater allowed for effective and temporary control of its optical characteristics, which made dura mater more transparent, increased the ability of light penetrating the tissue, and consequently improved the optical imaging depth. It is a significant study, which can improve penetration of optical imaging of cerebral function and acquire more information of the deep brain tissue.

Discovery of a Small Molecule Probe That Post-Translationally Stabilizes the Survival Motor Neuron Protein for the Treatment of Spinal Muscular Atrophy.

PubMed

Rietz, Anne; Li, Hongxia; Quist, Kevin M; Cherry, Jonathan J; Lorson, Christian L; Burnett, Barrington G; Kern, Nicholas L; Calder, Alyssa N; Fritsche, Melanie; Lusic, Hrvoje; Boaler, Patrick J; Choi, Sungwoon; Xing, Xuechao; Glicksman, Marcie A; Cuny, Gregory D; Androphy, Elliot J; Hodgetts, Kevin J

2017-06-08

Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. We previously developed a high-throughput assay that employs an SMN2-luciferase reporter allowing identification of compounds that act transcriptionally, enhance exon recognition, or stabilize the SMN protein. We describe optimization and characterization of an analog suitable for in vivo testing. Initially, we identified analog 4m that had good in vitro properties but low plasma and brain exposure in a mouse PK experiment due to short plasma stability; this was overcome by reversing the amide bond and changing the heterocycle. Thiazole 27 showed excellent in vitro properties and a promising mouse PK profile, making it suitable for in vivo testing. This series post-translationally stabilizes the SMN protein, unrelated to global proteasome or autophagy inhibition, revealing a novel therapeutic mechanism that should complement other modalities for treatment of SMA.

Analysis of the optical characteristics of adipose tissue in vitro sensitized by indocyanine green and exposed to IR-laser irradiation

NASA Astrophysics Data System (ADS)

Yanina, I. Yu.; Doubrovski, V. A.; Tuchin, V. V.

2015-03-01

The effect of IR-laser irradiation on adipose tissue sensitized by indocyanine green is studied in vitro. Experiments and statistical analysis are used to show that wavelength-selective irradiation leads to an increase in the homogeneity of optical images of adipose cells with time. The transmission coefficient that is averaged over the image area weakly depends on the observation time. An increase in the homogeneity of images is interpreted as a result of immersion of optical inhomogeneities of tissue owing to the intracellular liquid that is released through the photochemically induced pores in cellular membranes. An increase in the optical homogeneity of the medium is compensated for by a decrease in the transmission coefficient of the sensitizer, which is manifested as a weak time dependence of the image-averaged transmittance of tissue.

Inhibition of MAO by fractions and constituents of hypericum extract.

PubMed

Bladt, S; Wagner, H

1994-10-01

The inhibition of monoamine oxidase (MAO) by six fractions from hypericum extract and three characteristic constituents (as pure substances) were analyzed in vitro and ex vivo to study the antidepressive mechanism of action. Rat brain homogenates were used as the in vitro model, while the ex vivo analysis was performed after intraperitoneal application of the test substances to albino rats. Massive inhibition of MAO-A could be shown with the total extract and all fractions only at the concentration of 10(-3) mol/L. At 10(-4) mol/L, one fraction rich in flavonoides showed an inhibition of 39%, and all other fractions demonstrated less than 25% inhibition. Using pure hypericin as well as in all ex vivo experiments, no relevant inhibiting effects could be shown. From the results it can be concluded that the clinically proven antidepressive effect of hypericum extract cannot be explained in terms of MAO inhibition.

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

PubMed

Woodman, P G; Adamczewski, J P; Hunt, T; Warren, G

1993-05-01

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

Pinosylvin-Based Polymers: Biodegradable Poly(Anhydride-Esters) for Extended Release of Antibacterial Pinosylvin.

PubMed

Bien-Aime, Stephan; Yu, Weiling; Uhrich, Kathryn E

2016-07-01

Pinosylvin is a natural stilbenoid known to exhibit antibacterial bioactivity against foodborne bacteria. In this work, pinosylvin is chemically incorporated into a poly(anhydride-ester) (PAE) backbone via melt-condensation polymerization, and characterized with respect to its physicochemical and thermal properties. In vitro release studies demonstrate that pinosylvin-based PAEs hydrolytically degrade over 40 d to release pinosylvin. Pseudo-first order kinetic experiments on model compounds, butyric anhydride and 3-butylstilbene ester, indicate that the anhydride linkages hydrolyze first, followed by the ester bonds to ultimately release pinosylvin. An antibacterial assay shows that the released pinosylvin exhibit bioactivity, while in vitro cytocompatibility studies demonstrate that the polymer is noncytotoxic toward fibroblasts. These preliminary findings suggest that the pinosylvin-based PAEs can serve as food preservatives in food packaging materials by safely providing antibacterial bioactivity over extended time periods. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hyaluronic acid as a potential boron carrier for BNCT: Preliminary evaluation.

PubMed

Zaboronok, A; Yamamoto, T; Nakai, K; Yoshida, F; Uspenskii, S; Selyanin, M; Zelenetskii, A; Matsumura, Akira

2015-12-01

Hyaluronic acid (HA), a nonimmunogenic, biocompatible polymer found in different biological tissues, has the potential to attach to CD44 receptors on the surface of certain cancer cells, where the receptor is overexpressed compared with normal cells. Boron-hyaluronic acid (BHA) was tested for its feasibility as a potential agent for BNCT. BHA with low-viscosity 30 kDa HA could be administered by intravenous injection. The compound showed a certain degree of cytotoxicity and accumulation in C6 rat glioma cells in vitro. Instability of the chelate bonds between boron and HA and/or insufficient specificity of CD44 receptors on C6 cells to BHA could account for the insufficient in vitro accumulation. To ensure the future eligibility of BHA for BNCT experiments, using alternative tumor cell lines and chemically securing the chelate bonds or synthesizing BHA with boron covalently attached to HA might be required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of fluorescent lighting on in vitro micropropagation of Lemna minor

NASA Astrophysics Data System (ADS)

Somsri, Kollawat; Pinyopich, Pataradawn; Mohammed, Waleed S.

2010-05-01

The vegetative in vitro propagation of Lemna minor stain SING-4 exposed to two different types of fluorescent light sources, Philips TLD 36W/54 and Toshiba FL40T8BRF/36, was studied. The liquid culture medium contained 4.43gl-1 phytohormone-free full-strength Murashige & Skoog (MS) basal medium with vitamins, 30gl-1 sucrose, and 1gl-1 MES. The results showed that both plant cultures had undergone normal asexual reproduction with an exponential increase trend. Cultures exposed to Toshiba FL40T8BRF/36 reproduced at a slightly faster rate while expressing significantly greener foliage (leaf color chart shade No.8), which indicates the presence of more chlorophyll, than cultures exposed to Philips TLD 36W/54 (leaf color chart shade No.4). The data obtained from our experiment reveals that light emitted from Toshiba FL40T8BRF/36 produces healthier and higher quality cultures.

Dual-Responsive Molecular Probe for Tumor Targeted Imaging and Photodynamic Therapy

PubMed Central

Meng, Xiaoqing; Yang, Yueting; Zhou, Lihua; Zhang, li; Lv, Yalin; Li, Sanpeng; Wu, Yayun; Zheng, Mingbin; Li, Wenjun; Gao, Guanhui; Deng, Guanjun; Jiang, Tao; Ni, Dapeng; Gong, Ping; Cai, Lintao

2017-01-01

The precision oncology significantly relies on the development of multifunctional agents to integrate tumor targeting, imaging and therapeutics. In this study, a first small-molecule theranostic probe, RhoSSCy is constructed by conjugating 5′-carboxyrhodamines (Rho) and heptamethine cyanine IR765 (Cy) using a reducible disulfide linker and pH tunable amino-group to realize thiols/pH dual sensing. In vitro experiments verify that RhoSSCy is highly sensitive for quantitative analysis and imaging intracellular pH gradient and biothiols. Furthermore, RhoSSCy shows superb tumor targeted dual-modal imaging via near-infrared fluorescence (NIRF) and photoacoustic (PA). Importantly, RhoSSCy also induces strongly reactive oxygen species for tumor photodynamic therapy (PDT) with robust antitumor activity both in vitro and in vivo. Such versatile small-molecule theranostic probe may be promising for tumor targeted imaging and precision therapy. PMID:28638467

Synthesis and crystal structure determination of copper(II)-complex: In vitro DNA and HSA binding, pBR322 plasmid cleavage, cell imaging and cytotoxic studies.

PubMed

Tabassum, Sartaj; Zaki, Mehvash; Ahmad, Musheer; Afzal, Mohd; Srivastav, Saurabh; Srikrishna, Saripella; Arjmand, Farukh

2014-08-18

New Cu(II) complex 1 of indole-3-propionic acid and 1,10-phenanthroline was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. In vitro DNA binding studies of 1 was performed by employing UV-vis and fluorescence spectroscopic techniques. The binding affinity towards human serum albumin (HSA) was also investigated to understand the carrier role in body system, as the time dependent HPLC experiment of 1 revealed that bonded drug with protein releases slowly in presence of DNA. Complex 1 exhibited good anti-tumor activity (GI50 values <10 μg/ml), and to elucidate the mechanism of tumor inhibition, topoisomerase I enzymatic activity was carried out and further validated by cell imaging studies which clearly showed its nuclear localization. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

LOW PRESSURE ULTRAVEIOLET STUDIES FOR INACTIVATION OF GIARDIA MURIS CYSTS

EPA Science Inventory

Cysts of Giardia muris were inactivated using a low pressure ultravolet (UV) light source. Cyst viability was detemined by both in vitro excystation and animal infectivity. Cyst doeses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excy...

LOW PRESSURE ULTRAVIOLET STUDIES FOR INACTIVATION OF GIARDIA MURIS CYSTS

EPA Science Inventory

Cysts of Giardia muris were inactivated using a low pressure ultravolet (UV) light source. Cyst viability was detemined by both in vitro excystation and animal infectivity. Cyst doeses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excy...

Arsenic Metabolism by Human Gut Microbiota upon in Vitro Digestion of Contaminated Soils

EPA Science Inventory

Speciation analysis is essential when evaluating risks from, arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with animal microbiota. Howeve...

Effect of isopropyl myristate on the viscoelasticity and drug release of a drug-in-adhesive transdermal patch containing blonanserinEffect of isopropyl myristate on the viscoelasticity and drug release of a drug-in-adhesive transdermal patch containing blonanserinretain-->.

PubMed

Zhao, Chunyi; Quan, Peng; Liu, Chao; Li, Qiaoyun; Fang, Liang

2016-11-01

The purpose of this study was to investigate the effect of isopropyl myristate (IPM), a penetration enhancer, on the viscoelasticity and drug release of a drug-in-adhesive transdermal patch containing blonanserin. The patches were prepared with DURO-TAK ® 87-2287 as a pressure-sensitive adhesive (PSA) containing 5% ( w / w ) of blonanserin and different concentrations of IPM. An in vitro release experiment was performed and the adhesive performance of the drug-in-adhesive patches with different concentrations of IPM was evaluated by a rolling ball tack test and a shear-adhesion test. The glass transition temperature ( T g ) and rheological parameters of the drug-in-adhesive layers were determined to study the effect of IPM on the mechanical properties of the PSA. The results of the in vitro release experiment showed that the release rate of blonanserin increased with an increasing concentration of IPM. The rolling ball tack test and shear-adhesion test showed decreasing values with increasing IPM concentration. The results were interpreted on the basis of the IPM-induced plasticization of the PSA, as evidenced by a depression of the glass transition temperature and a decrease in the elastic modulus. In conclusion, IPM acted as a plasticizer on DURO-TAK ® 87-2287, and it increased the release of blonanserin and affected the adhesive properties of the PSA.

Fiber optic laser light scattering measurement of ciliary function of the fallopian tube

NASA Astrophysics Data System (ADS)

Halbert, Sheridan A.; Lim, Kap; Lee, Wylie I.

1990-07-01

A fiber-optic laser light-scattering system (FLS) for measuring ciliary function was evaluated by means of three sets of in vitro experiments. First, FLS performance was compared to that of a previously proven benchtop laser system (BLS). Using tissue excised from rabbit fallopian tubes, ciliary beat frequency (CBF) of each sample was measured with FLS and BLS. Paired CBF measurements showed excellent correlation between the two systems (r =0.93). Second, the FLS was used to evaluate the dependency of CBF on temperature (T) by using tissue sampies of rabbit oviductal fimbna. Regression analysis of CBF vs T showed a linear relationship over the range of 18-37°C for both individual samples (r =0.98) and pooled data from all experiments (r = 0.84). Fmally, the relalionship between CBF and ciliary ovum transport rate (TR) was tested by using T to modulate CBF of rabbit fimbria, in vitro. The relationship was linear over the range of CBF from 10 to 30 Hz (r2 = 0.83). At 37°C, CBF = 31+/-1 Hz, and TR = O.12+/-.02 mm/sec. equal to ovum transport rate in situ. The FLS is a valuable tool for characterizing ciliary activity and thus ovum transport function. Owing to the fact that ciliary dyskinesia resulting from disease of the fallopian tube is associated with infeitility, the FLS may be useful to acquire data important to the clinical evaluation of fallopian tube function and female infertility.

Effect of P2X7 Receptor Knockout on AQP-5 Expression of Type I Alveolar Epithelial Cells

PubMed Central

Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

2014-01-01

P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

1996-08-01

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Evolution of the Pseudomonas aeruginosa Aminoglycoside Mutational Resistome In Vitro and in the Cystic Fibrosis Setting.

PubMed

López-Causapé, Carla; Rubio, Rosa; Cabot, Gabriel; Oliver, Antonio

2018-04-01

Inhaled administration of high doses of aminoglycosides is a key maintenance treatment of Pseudomonas aeruginosa chronic respiratory infections in cystic fibrosis (CF). We analyzed the dynamics and mechanisms of stepwise high-level tobramycin resistance development in vitro and compared the results with those of isogenic pairs of susceptible and resistant clinical isolates. Resistance development correlated with fusA1 mutations in vitro and in vivo. pmrB mutations, conferring polymyxin resistance, were also frequently selected in vitro In contrast, mutational overexpression of MexXY, a hallmark of aminoglycoside resistance in CF, was not observed in in vitro evolution experiments. Copyright © 2018 American Society for Microbiology.

Evaluation of sources of variation on in vitro fermentation kinetics of feedstuffs in a gas production system.

PubMed

Keim, Juan P; Alvarado-Gilis, Christian; Arias, Rodrigo A; Gandarillas, Mónica; Cabanilla, Jaime

2017-10-01

The aim of this study was to evaluate the effect of different sources of variation in gas production technique on the in vitro gas production kinetics of feedstuffs. Triplicates of commercial concentrate, grass silage, grass hay and grass pasture were incubated in three experiments: experiment 1 assessed two agitation methods; experiment 2 evaluated different rumen inocula (pooled or different donor cows for each incubation run); and experiment 3 used Goering-Van Soest or Mould buffers for media preparation. Gas production data were fitted into the Michaelis-Menten model and then subjected to analysis of variance. Gas production (GP) at 48 h and asymptote gas production (A) were lower when bottles were continuously under horizontal movement. Time to produce half and 75% of A, and A were affected by rumen inocula, while buffer type affected time to produce half and 25% of A and GP. No interactions between substrates and sources of variation were observed, suggesting that the effects of substrates on GP parameters were not modified. It is concluded that comparison of numerical data from in vitro experiments that follow different protocols must be done carefully. However, the ranking of different substrates is more robust and less affected by the sources of variation. © 2017 Japanese Society of Animal Science.

In Silico Reconstitution of Actin-Based Symmetry Breaking and Motility

PubMed Central

Dayel, Mark J.; Akin, Orkun; Landeryou, Mark; Risca, Viviana; Mogilner, Alex; Mullins, R. Dyche

2009-01-01

Eukaryotic cells assemble viscoelastic networks of crosslinked actin filaments to control their shape, mechanical properties, and motility. One important class of actin network is nucleated by the Arp2/3 complex and drives both membrane protrusion at the leading edge of motile cells and intracellular motility of pathogens such as Listeria monocytogenes. These networks can be reconstituted in vitro from purified components to drive the motility of spherical micron-sized beads. An Elastic Gel model has been successful in explaining how these networks break symmetry, but how they produce directed motile force has been less clear. We have combined numerical simulations with in vitro experiments to reconstitute the behavior of these motile actin networks in silico using an Accumulative Particle-Spring (APS) model that builds on the Elastic Gel model, and demonstrates simple intuitive mechanisms for both symmetry breaking and sustained motility. The APS model explains observed transitions between smooth and pulsatile motion as well as subtle variations in network architecture caused by differences in geometry and conditions. Our findings also explain sideways symmetry breaking and motility of elongated beads, and show that elastic recoil, though important for symmetry breaking and pulsatile motion, is not necessary for smooth directional motility. The APS model demonstrates how a small number of viscoelastic network parameters and construction rules suffice to recapture the complex behavior of motile actin networks. The fact that the model not only mirrors our in vitro observations, but also makes novel predictions that we confirm by experiment, suggests that the model captures much of the essence of actin-based motility in this system. PMID:19771152

In vitro investigation of anodization and CaP deposited titanium surface using MG63 osteoblast-like cells

NASA Astrophysics Data System (ADS)

Lee, J. M.; Lee, J. I.; Lim, Y. J.

2010-03-01

The aim of the present study was to investigate surface characteristics in four different titanium surfaces (AN: anodized at 270 V; AN-CaP: anodic oxidation and CaP deposited; SLA: sandblasted and acid etched; MA: machined) and to evaluate biological behaviors such as cell adhesion, cell proliferation, cytoskeletal organization, and osteogenic protein expression of MG63 osteoblast-like cells at the early stage. Surface analysis was performed using scanning electron microscopy, thin-film X-ray diffractometry, and a confocal laser scanning microscope. In order to evaluate cellular responses, MG63 osteoblast-like cells were used. The cell viability was evaluated by MTT assay. Immunofluorescent analyses of actin, type I collagen, osteonectin and osteocalcin were performed. The anodized and CaP deposited specimen showed homogeneously distributed CaP particles around micropores and exhibited anatase type oxides, titanium, and HA crystalline structures. This experiment suggests that CaP particles on the anodic oxidation surface affect cellular attachment and spreading. When designing an in vitro biological study for CaP coated titanium, it must be taken into account that preincubation in medium prior to cell seeding and the cell culture medium may affect the CaP coatings. All these observations illustrate the importance of the experimental conditions and the physicochemical parameters of the CaP coating. It is considered that further evaluations such as long-term in vitro cellular assays and in vivo experiments should be necessary to figure out the effect of CaP deposition to biological responses.

In planta transformation method for T-DNA transfer in orchids

NASA Astrophysics Data System (ADS)

Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko

2014-03-01

Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro∷PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro∷GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.

Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.

PubMed

George, F; Vrancken, M; Verhaeghe, B; Verhoeye, F; Schneider, Y-J; Massip, A; Donnay, I

2006-09-15

Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrations<18 mg/mL on in vitro bovine blastocysts. Increasing concentrations of peptones were then added in the freezing medium. The surviving and hatching rates were not affected by comparison with those observed with BSA. No significant difference was observed between groups either for the total number of cells or for the ratio ICM/Total cell, nor for the rate of apoptosis in surviving embryos. When embryos were cryopreserved in 1.8 mg/mL peptone, the hatching rate and embryo quality as assessed at 48 h post-thawing were not significantly different from those of unfrozen embryos. In a second experiment two additives were added in this animal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

Comparison of two different in vivo models and an in vitro model for caloric determination of four novel fiber ingredients.

PubMed

Cervantes-Pahm, Sarah; Knapp, Brenda K; Kim, Beob G; Liu, Yanhong; Parsons, Carl M; Fahey, George C; Stein, Hans H

2013-12-18

The objective of this study was to compare two in vivo methods using pigs and roosters and an in vitro method for determining the caloric value of four fiber sources [i.e., two resistant starches (RS 60 and RS 75), soluble corn fiber (SCF 70), and pullulan]. Metabolizable energy (ME) in pigs and true metabolizable energy (TMEn) in roosters were determined by using 72 barrows and 24 roosters, respectively. A two-step in vitro procedure was used to quantify monosaccharides released. Results of the two in vivo experiments corresponded well with RS 75 having the least caloric value (7.55 MJ/kg in pigs; 6.19 MJ/kg in roosters) and pullulan having the greatest caloric value (12.21 MJ/kg in pigs; 13.94 MJ/kg in roosters). The caloric values for all the fiber ingredients were less (P < 0.05) than in MD both in pigs and in roosters. Despite some limitations, results of the in vitro procedure corresponded well with the in vivo experiments where the concentration of glucose hydrolyzed from RS 60, RS 75, and SCF 70, but not pullulan, was less (P < 0.05) than the concentration of glucose hydrolyzed from MD. However, the greatest accuracy was obtained in the in vivo experiments.

Myocardial temperature distribution under cw Nd:YAG laser irradiation in in vitro and in vivo situations: theory and experiment

NASA Astrophysics Data System (ADS)

Splinter, Robert; Littmann, Laszlo; Tuntelder, Jan R.; Svenson, Robert H.; Chuang, Chi Hui; Tatsis, George P.; Semenov, Serguei Y.; Nanney, Glenn A.

1995-01-01

Tissue samples ranging from 2 to 16 mm in thickness were irradiated at 1064 nm with energies ranging from 40 to 2400 J. Coagulation lesions of in vitro and in vivo experiments were subjected to temperature profiling and submitted for histology. Irreversible damage was calculated with the damage integral formalism, following the bioheat equation solved with Monte Carlo computer light-distribution simula-tions. Numerical temperature rise and coagulation depth compared well with the in vitro results. The in vivo data required a change in the optical properties based on integrating sphere measurements for high irradiance to make the experimental and numerical data converge. The computer model has successfully solved several light-tissue interaction situations in which scattering dominates over absorption.

A NOVEL WEARABLE PUMP-LUNG DEVICE: IN-VITRO AND ACUTE IN-VIVO STUDY

PubMed Central

Zhang, Tao; Wei, Xufeng; Bianchi, Giacomo; Wong, Philip M.; Biancucci, Brian; Griffith, Bartley P.; Wu, Zhongjun J.

2011-01-01

Background To provide long-term ambulatory cardiopulmonary and respiratory support for adult patients, a novel wearable artificial pump-lung device has been developed. The design features, in-vitro and acute in-vivo performance of this device are reported in this paper. Methods This device features a uniquely designed hollow fiber membrane bundle integrated with a magnetically levitated impeller together to form one ultra-compact pump-lung device, which can be placed like current paracorporeal ventricular assist devices to allow ambulatory support. The device is 117 mm in length and 89 mm in diameter and has a priming volume of 115 ml. In-vitro hydrodynamic, gas transfer and biocompatibility experiments were carried out in mock flow loops using ovine blood. Acute in-vivo characterization was conducted in ovine by surgically implanting the device between right atrium and pulmonary artery. Results The in-vitro results showed that the device with a membrane surface area of 0.8 m2 was capable of pumping blood from 1 to 4 L/min against a wide range of pressures and transferring oxygen at a rate of up to 180 ml/min at a blood flow of 3.5 L/min. Standard hemolysis tests demonstrated low hemolysis at the targeted operating condition. The acute in-vivo results also confirmed that the device can provide sufficient oxygen transfer with excellent biocompatibility. Conclusions Base on the in-vitro and acute in-vivo study, this highly integrated wearable pump-lung device can provide efficient respiratory support with good biocompatibility and it is ready for long-term evaluation. PMID:22014451

THE ROLE OF LYMPHOCYTES IN THE SENSITIZATION OF RATS TO RENAL HOMOGRAFTS

PubMed Central

Strober, S.; Gowans, J. L.

1965-01-01

In order to study the role of blood-borne small lymphocytes in the sensitization of rats to renal homografts 2 techniques for the perfusion of isolated rat kidneys were employed: (a) the in vitro perfusion of kidneys with thoracic duct cells suspended in either an artificial medium or in blood; the perfusates were then injected into rats syngeneic with the lymphocyte donors; (b) the in vivo perfusion of kidneys with blood issuing from the femoral artery and returning to the femoral vein of living rats. The degree of sensitization conferred on the recipients by the perfusates was assessed by applying a skin homograft from the kidney donor and scoring the epithelial necrosis at 6 days. The in vitro experiments indicated that parental strain thoracic duct cells, which had passed through an F1 hybrid kidney could confer upon a parental rat sensitivity to an F1 skin graft. Several perfusions with radioactively labelled lymphocytes showed that the injected cells migrated to the lymph nodes and spleen of the recipients Labelled large pyroninophilic cells were occasionally seen in the spleen and lymph nodes of recipients, and it was suggested that these had arisen from the injected cells. Although the in vitro perfusions with blood indicated that renal homografts might sensitize their hosts within 1 hour, the in vivo perfusions suggested that about 5 to 12 hours were required. The more rapid sensitization in vitro was possibly due to the more frequent opportunity for contact between lymphocytes and kidney vascular endothelium which was afforded by the conditions in vitro. PMID:14316949

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-polymerizing activity.

PubMed

Tajima, Kenji; Han, Xuerong; Satoh, Yasuharu; Ishii, Ayako; Araki, Yuji; Munekata, Masanobu; Taguchi, Seiichi

2012-04-01

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).

Arsenic Metabolism by Human Gut Microbiota upon In Vitro Digestion of Contaminated Soils

EPA Science Inventory

Background: Speciation analysis is essential when evaluating risks from arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with ...

Influence of fluoride additions on biological and mechanical properties of Na2O-CaO-SiO2-P2O5 glass-ceramics.

PubMed

Li, H C; Wang, D G; Hu, J H; Chen, C Z

2014-02-01

Two series of Na2O-CaO-SiO2-P2O5 glass-ceramics doped with NH4HF2 (G-NH4HF2) or CaF2 (G-CaF2) have been prepared by sol-gel method. The glass-ceramic phase composition and morphology were characterized by X-ray diffraction (XRD) and scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS). The mechanical properties and thermal expansion coefficient were measured by a microhardness tester, an electronic tensile machine and a thermal expansion coefficient tester. The structure difference between these two glass-ceramics was investigated by Fourier transform infrared spectroscopy (FTIR), and the in vitro bioactivity of the glass-ceramics was determined by in vitro simulated body fluid (SBF) immersion test. The hemolysis test, in vitro cytotoxicity test, systemic toxicity test and the implanted experiment in animals were used to evaluate the biocompatibility of the glass-ceramics. The mechanical properties of sample G-NH4HF2 are lower than that of sample G-CaF2, and the bioactivity of sample G-NH4HF2 is better than that of sample G-CaF2. The thermal expansion coefficients of these two glass-ceramics are all closer to that of Ti6Al4V. After 7 days of SBF immersion, apatites were induced on glass-ceramic surface, indicating that the glass-ceramics have bioactivity. The hemolysis test, in vitro cytotoxicity test and systemic toxicity test demonstrate that the glass-ceramics do not cause hemolysis reaction, and have no toxicity to cell and living animal. The implanted experiment in animals shows that bone tissue can form a good osseointegration with the implant after implantation for two months, indicating that the glass-ceramics are safe to serve as implants. Copyright © 2013 Elsevier B.V. All rights reserved.

The Most Effective Gold Nanorod Size for Plasmonic Photothermal Therapy: Theory and In Vitro Experiments

PubMed Central

2015-01-01

The development of new and improved photothermal contrast agents for the successful treatment of cancer (or other diseases) via plasmonic photothermal therapy (PPTT) is a crucial part of the application of nanotechnology in medicine. Gold nanorods (AuNRs) have been found to be the most effective photothermal contrast agents, both in vitro and in vivo. Therefore, determining the optimum AuNR size needed for applications in PPTT is of great interest. In the present work, we utilized theoretical calculations as well as experimental techniques in vitro to determine this optimum AuNR size by comparing plasmonic properties and the efficacy as photothermal contrast agents of three different sizes of AuNRs. Our theoretical calculations showed that the contribution of absorbance to the total extinction, the electric field, and the distance at which this field extends away from the nanoparticle surface all govern the effectiveness of the amount of heat these particles generate upon NIR laser irradiation. Comparing between three different AuNRs (38 × 11, 28 × 8, and 17 × 5 nm), we determined that the 28 × 8 nm AuNR is the most effective in plasmonic photothermal heat generation. These results encouraged us to carry out in vitro experiments to compare the PPTT efficacy of the different sized AuNRs. The 28 × 8 nm AuNR was found to be the most effective photothermal contrast agent for PPTT of human oral squamous cell carcinoma. This size AuNR has the best compromise between the total amount of light absorbed and the fraction of which is converted to heat. In addition, the distance at which the electric field extends from the particle surface is most ideal for this size AuNR, as it is sufficient to allow for coupling between the fields of adjacent particles in solution (i.e., particle aggregates), resulting in effective heating in solution. PMID:24433049

[Isolation and in vitro metabolic characterization of a lactate-utilizing bacterium from goat rumen].

PubMed

Long, Liming; Mao, Shengyong; Su, Yong; Zhu, Weiyun

2008-12-01

A lactate-utilizing, propionate-producing bacterium, strain L9, was isolated from rumen of goat fed with high concentrate by utilizing modified Hungate technique and anaerobic culture technique. The effect of the strain L9 culture on the rumen fermentation was further studied. According to the characteristics of morphology, physiology, biochemistry tests and sequence comparison of 16S rRNA gene, strain L9 was identified as selenomonas ruminantium. The influence of strain L9 culture on in vitro rumen fermentation was studied using mixed rumen micro-organisms of goats as inoculums. The results of the metabolism experiment showed that it was capable of using lactate as the sole carbon source, and 90 mmol/L lactate in LH medium could be completely utilized after 24 h incubation. As compared with the control, strain L9 culture addition significantly increased the total volatile fatty acids (TVFA), the percentage of propionate and pH value, while reduced the ratio of acetate to propionate and lactate production (P < 0.05). The results suggested that strain L9 can reduce lactic acid production and enhance the TVFA and propionate production in in vitro fermentation, and thus could be beneficial for the fermentation of rumen microorganisms.

Photodynamic Therapy Combined with Terbinafine Against Chromoblastomycosis and the Effect of PDT on Fonsecaea monophora In Vitro

PubMed Central

Hu, Yongxuan; Huang, Xiaowen; Lu, Sha; Hamblin, Michael R.; Mylonakis, Eleftherios; Zhang, Junmin

2014-01-01

Chromoblastomycosis, a chronic fungal infection of skin and subcutaneous tissue caused by dematiaceous fungi, is associated with low cure and high relapse rates. Among all factors affecting clinical outcome, etiological agents have an important position. In southern China, Fonsecaea pedrosoi and Fonsecaea monophora are main causative agents causing Chromoblastomycosis. We treated one case of chromoblastomycosis by photodynamic therapy (PDT) of 5-aminolevulinic acid (ALA) irradiation combined with terbinafine 250 mg a day. The lesions were improved after two sessions of ALA-PDT treatment, each including nine times, at an interval of 1 week, combined with terbinafine 250 mg/day oral, and clinical improvement could be observed. In the following study, based on the clinical treatment, the effect of PDT and antifungal drugs on this isolate was detected in vitro. It showed sensitivity to terbinafine, itraconazole or voriconazole, and PDT inhibited the growth. Both the clinic and experiments in vitro confirm the good outcome of ALA-PDT applied in the inhibition of F. monophora. It demonstrated that combination of antifungal drugs with ALA-PDT arises as a promising alternative method for the treatment of these refractory cases of chromoblastomycosis. PMID:25366276

Evaluation of the effect of hydroxypropyl-β-cyclodextrin on topical administration of milk thistle extract.

PubMed

Spada, Gianpiera; Gavini, Elisabetta; Cossu, Massimo; Rassu, Giovanna; Carta, Antonio; Giunchedi, Paolo

2013-01-30

Two water in oil emulsions composed by eudermic ingredients as glycerin, cocoa butter, almond oil and a variety of lipids, were enriched respectively with milk thistle dry extract (MT) or with a binary complex composed by MT and hydroxypropyl-β-cyclodextrin (HP) (1:4 w/w) correspondent to 1% (w/w) in sylimarine in order to obtain two different emulsions designed for the skin delivery and determine influence of hydroxypropyl-β-cyclodextrin on the extract delivery and permeation. Uv-vis spectrophotometric analyses demonstrated that phytocomplex formation influences the finding of MT after the complexation process and the in vitro antioxidant activity. Further in vitro and ex vivo experiments demonstrated that the penetration capability of MT from formulations is strictly influenced by the phytocomplex able to control MT permeation; moreover phytocomplex increases flavonoids stability during the in vitro tests. Additionally, in vivo studies showed that the penetration into the stratum corneum of the active ingredients is effectively achieved by the phytocomplex formation, in fact about 80% of MT is absorbed by the skin along 1h despite the 30% of MT not complexed absorbed during the same period. Copyright © 2012 Elsevier Ltd. All rights reserved.

Photothermal and biodegradable polyaniline/porous silicon hybrid nanocomposites as drug carriers for combined chemo-photothermal therapy of cancer.

PubMed

Xia, Bing; Wang, Bin; Shi, Jisen; Zhang, Yu; Zhang, Qi; Chen, Zhenyu; Li, Jiachen

2017-03-15

To develop photothermal and biodegradable nanocarriers for combined chemo-photothermal therapy of cancer, polyaniline/porous silicon hybrid nanocomposites had been successfully fabricated via surface initiated polymerization of aniline onto porous silicon nanoparticles in our experiments. As-prepared polyaniline/porous silicon nanocomposites could be well dispersed in aqueous solution without any extra hydrophilic surface coatings, and showed a robust photothermal effect under near-infrared (NIR) laser irradiation. Especially, after an intravenous injection into mice, these biodegradable porous silicon-based nanocomposites as non-toxic agents could be completely cleared in body. Moreover, these polyaniline/porous silicon nanocomposites as drug carriers also exhibited an efficient loading and dual pH/NIR light-triggered release of doxorubicin hydrochloride (DOX, a model anticancer drug). Most importantly, assisted with NIR laser irradiation, polyaniline/PSiNPs nanocomposites with loading DOX showed a remarkable synergistic anticancer effect combining chemotherapy with photothermal therapy, whether in vitro or in vivo. Therefore, based on biodegradable PSiNPs-based nanocomposites, this combination approach of chemo-photothermal therapy would have enormous potential on clinical cancer treatments in the future. Considering the non-biodegradable nature and potential long-term toxicity concerns of photothermal nanoagents, it is of great interest and importance to develop biodegradable and photothermal nanoparticles with an excellent biocompatibility for their future clinical applications. In our experiments, we fabricated porous silicon-based hybrid nanocomposites via surface initiated polymerization of aniline, which showed an excellent photothermal effect, aqueous dispersibility, biodegradability and biocompatibility. Furthermore, after an efficient loading of DOX molecules, polyaniline/porous silicon nanocomposites exhibited the remarkable synergistic anticancer effect, whether in vitro and in vivo. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Monitoring radio-frequency thermal ablation with ultrasound by low frequency acoustic emissions--in vitro and in vivo study.

PubMed

Winkler, Itai; Adam, Dan

2011-05-01

The object of this study was to evaluate the monitoring of thermal ablation therapy by measuring the nonlinear response to ultrasound insonation at the region being treated. Previous reports have shown that during tissue heating, microbubbles are formed. Under the application of ultrasound, these microbubbles may be driven into nonlinear motion that produces acoustic emissions at sub-harmonic frequencies and a general increase of emissions at low frequencies. These low frequency emissions may be used to monitor ablation surgery. In this study, a modified commercial ultrasound system was used for transmitting ultrasound pulses and for recording raw RF-lines from a scan plane in porcine (in vitro) and rabbit (in vivo) livers during radio-frequency ablation (RFA). The transmission pulse was 15 cycles in length at 4 MHz (in vitro) and 3.6 MHz (in vivo). Thermocouples were used for monitoring temperatures during the RFA treatment.In the in vitro experiments, recorded RF signals (A-lines) were segmented, and the total energy was measured at two different frequency bands: at a low frequency band (LFB) of 1-2.5 MHz and at the transmission frequency band (TFB) of 3.5-4.5 MHz. The mean energy at the LFB and at the TFB increased substantially in areas adjacent to the RF needle. These energies also changed abruptly at higher temperatures, thus, producing great variance in the received energy. Mean energies in areas distant from RF needle showed little change and variation during treatment. It was also shown that a 3 dB increase of energy at the low frequency band was typically obtained in regions in which temperature was above 53.3 ± 5° C. Thus, this may help in evaluating regions undergoing hyperthermia. In the in vivo experiments, an imaging algorithm based on measuring the LFB energy was used. The algorithm performs a moving average of the LFB energies measured at segments within the scan plane.Results show that a colored region is formed on the image and that it is similar in size to a measurement of the lesion from gross pathology, with a correlation coefficient of 0.743. Copyright © 2011. Published by Elsevier Inc.

Prediction of utilizable true protein of mixed rations for sheep using an in vitro incubation technique.

PubMed

Li, Yuan-Xiao; Zhao, Guang-Yong

2007-04-01

The objective of the present experiment was to study the relationship between in vitro utilizable true protein (uTP) and in vivo-uTP of sheep rations by regression analysis. A further aim was to analyse if in vivo-uTP of mixed rations could be predicted by regression analysis between in vitro-uTP and in vivo-uTP, using N-retention of sheep as important evaluation criteria of protein value. Three adult male sheep (body weight [BW] 46 + 1.3 kg) fitted with rumen cannulas and simple T-type duodenal cannulas were fed with twelve typical rations with graded levels of crude protein and true protein in four experiments according a 3 x 3 Latin square design. Each experimental period included an adaptation (7 days), a N balance trial (4 days) and a collection of duodenal digesta (3 days). During collection of duodenal digesta, polyethylene glycol and chromium oxide were used as dual markers for the measurement of duodenal digesta flow and calculation of the in vivo-uTP of duodenal digesta. The in vitro-uTP of the rations was determined using the in vitro incubation technique of Zhao and Lebzien (2000). It was found that both in vitro-uTP intake and in vivo-uTP intake were significantly correlated with N-retention (p < 0.001) and that there was a significant linear relationship between the content of in vitro-uTP and in vivo-uTP in rations (p < 0.001). Therefore, it was concluded that the used in vitro incubation technique is suitable for the determination of in vitro-uTP of mixed rations for sheep, and that the amount of in vivo-uTP can be predicted by regression between in vitro-uTP and in vivo-uTP.

[Effect of vitamins B1, B2, B6, folic acid and vitamin C on the motor activity of chicken's intestines in chronic experiments and in vitro].

PubMed

Nagórna-Stasiak, B; Wawrzeńska, M

1987-01-01

The studies were carried out on 33 chickens of the broiler breed in chronic experiments and in vitro. In the chronic experiments the motility of the jejunum under the influence of vitamins of group B and vitamine C was recorded in 8 chickens. The vitamins were used at concentrations from 10 mg/l to 2.5 x 10(3) mg/l. In the experiments in vitro, the motility of the isolated segment of the jejunum was recorded by the method of Magnus. In this part of experiments the chickens were divided into 3 groups, of which group I (15 chickens) were fed with DKA finischer mixture, group II (5 hens) received, besides the mixture, per os 200 mg of vitamin C for 2 weeks, group III (5 hens) received the mixture and for 2 weeks intraperitoneally 200 mg of vitamin C. The effect of vitamins of group B in vitro was determined in chickens of group I, whereas that of vitamin C in chickens of group I, II and III. At the same time the level of vitamin C in the wall of the jejunum was determined by the method of Roe-Kuenther. It was shown that vitamin B2 and folic acid caused stimulation of intestine motility in the chickens, while vitamin B1, B6 and C decreased the motoric activity. Increased level of vitamin C in the intestinal wall resulted in increased intestine sensitivity. Chicken intestines sensitivity to vitamins was 10 times stronger to vitamins than that of the intestines of rabbits.

Assessment of early combination effects of colistin and meropenem against Pseudomonas aeruginosa and Acinetobacter baumannii in dynamic time-kill experiments.

PubMed

Tängdén, Thomas; Karvanen, Matti; Friberg, Lena E; Odenholt, Inga; Cars, Otto

2017-07-01

In view of the paucity of clinical evidence, in vitro studies are needed to find antibiotic combinations effective against multidrug-resistant Gram-negative bacteria. Interpretation of in vitro effects is usually based on bacterial growth after 24 h in time-kill and checkerboard experiments. However, the clinical relevance of the effects observed in vitro is not established. In this study we explored alternative output parameters to assess the activities of colistin and meropenem against Pseudomonas aeruginosa and Acinetobacter baumannii. Four strains each of P. aeruginosa and A. baumannii were exposed to colistin and meropenem, alone and in combination, in 8 h dynamic time-kill experiments. Initial (1 h), maximum and 8 h bacterial reductions and the area under the bacterial time-kill curve were evaluated. Checkerboards, interpreted based on fractional inhibitory concentration indices after 24 h, were performed for comparison. In the time-kill experiments, the combination resulted in enhanced 1 h, maximum and 8 h bacterial reductions against 2, 3 and 5 of 8 strains, respectively, as compared to the single drugs. A statistically significant reduction in the area under the time-kill curve was observed for three strains. In contrast, the checkerboards did not identify synergy for any of the strains. Combination effects were frequently found with colistin and meropenem against P. aeruginosa and A. baumannii in time-kill experiments but were not detected with the checkerboard method. We propose that the early dynamics of bacterial killing and growth, which may be of great clinical importance, should be considered in future in vitro combination studies.

In vitro and in vivo delivery of the secretagogue diadenosine tetraphosphate from conventional and silicone hydrogel soft contact lenses

PubMed Central

Dominguez-Godinez, Carmen Olalla; Martin-Gil, Alba; Carracedo, Gonzalo; Guzman-Aranguez, Ana; González-Méijome, José Manuel; Pintor, Jesús

2013-01-01

Purpose To evaluate the possible use of soft contact lenses (CL) to improve the secretagogue role of diadenosine tetraphosphate (Ap4A) promoting tear secretion. Methods Two conventional hydrogel CL (Omafilcon A and Ocufilcon D) and two silicone hydrogel (SiH) CL (Comfilcon A and Balafilcon A) were used. Ap4A was loaded into the lenses by soaking in a 1 mM Ap4A solution during 12 h. In vitro experiments were performed by placing the lenses in multi-wells during 2 h containing 1 ml of ultrapure water. 100 μl aliquots were taken at time zero and every minute for the first 10 min, and then every 15 min. In vivo experiments were performed in New Zealand rabbits and both the dinucleotide release from SiH and tear secretion were measured by means of Schirmer strips and high-pressure liquid chromatography (HPLC) analysis. Results Ap4A in vitro release experiments in hydrogel CL presented a release time 50 (RT50) of 3.9 ± 0.2 min and 3.1 ± 0.1 min for the non-ionic and the ionic CL, respectively. SiH CL released also Ap4A with RT50 values of 5.1 ± 0.1 min for the non-ionic and 2.7 ± 0.1 min for the ionic CL. In vivo experiments with SiH CL showed RT50 values of 9.3 ± 0.2 min and 8.5 ± 0.2 min for the non-ionic and the ionic respectively. The non-ionic lens Ap4A release was able to induce tear secretion above baseline tear levels for almost 360 min. Conclusion The delivery of Ap4A is slower and the effect lasts longer with non-ionic lenses than ionic lenses.

In vitro and in vivo studies of biodegradable fine grained AZ31 magnesium alloy produced by equal channel angular pressing.

PubMed

Ratna Sunil, B; Sampath Kumar, T S; Chakkingal, Uday; Nandakumar, V; Doble, Mukesh; Devi Prasad, V; Raghunath, M

2016-02-01

The objective of the present work is to investigate the role of different grain sizes produced by equal channel angular pressing (ECAP) on the degradation behavior of magnesium alloy using in vitro and in vivo studies. Commercially available AZ31 magnesium alloy was selected and processed by ECAP at 300°C for up to four passes using route Bc. Grain refinement from a starting size of 46μm to a grain size distribution of 1-5μm was successfully achieved after the 4th pass. Wettability of ECAPed samples assessed by contact angle measurements was found to increase due to the fine grain structure. In vitro degradation and bioactivity of the samples studied by immersing in super saturated simulated body fluid (SBF 5×) showed rapid mineralization within 24h due to the increased wettability in fine grained AZ31 Mg alloy. Corrosion behavior of the samples assessed by weight loss and electrochemical tests conducted in SBF 5× clearly showed the prominent role of enhanced mineral deposition on ECAPed AZ31 Mg in controlling the abnormal degradation. Cytotoxicity studies by MTT colorimetric assay showed that all the samples are viable. Additionally, cell adhesion was excellent for ECAPed samples particularly for the 3rd and 4th pass samples. In vivo experiments conducted using New Zealand White rabbits clearly showed lower degradation rate for ECAPed sample compared with annealed AZ31 Mg alloy and all the samples showed biocompatibility and no health abnormalities were noticed in the animals after 60days of in vivo studies. These results suggest that the grain size plays an important role in degradation management of magnesium alloys and ECAP technique can be adopted to achieve fine grain structures for developing degradable magnesium alloys for biomedical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

[In vitro and ex vivo EPR investigation of metabolic changes in blood under the action of radiotoxins obtained from irradiated potato tubers].

PubMed

Ibragimova, M I; Petukhov, V Iu; Zheglov, E P; Koniukhov, G V; Nizamov, R N

2004-01-01

The effect of radiotoxin (RT) obtained from y-irradiated potato tubes on blood of sheep and mice has been investigated by using in vitro and ex vivo EPR. In experiments in vitro, the action of different preparations (RT, extract from unirradiated potato tubers, 1%-HCl or 30%-hydrogen peroxide) on sheep blood has been compared. It has been established that RT is an effective oxidant (like 1%-HCl) of haem iron that leads to an increase of the methemoglobin concentration. The specific peculiarity of RT effect on blood in vitro is an appearance of two well-resolved lines from methemoglobin belonging, probably, to different paramagnetic centers. The signal from nonspecific complexes of Fe3+ has been also observed. Ex vivo EPR spectra markedly differ from these obtained in experiments in vitro. An additional line with g approximately 2.005 and width 6 G in 30 minutes after intraperitoneal RT injection in the lethal dose (0.2 ml of preparation containing of 2 mg RT) has been revealed. Subsequent intoxication of mice is accompanied by the appearance of the signal from nitrosyl complexes in EPR spectra. These differences in experimental results of in vitro and ex vivo EPR can be explained by launch of compensatory adaptive response of organism on the action of highly toxic preparation.

Dopamine receptor D4 internalization requires a beta-arrestin and a visual arrestin.

PubMed

Deming, Janise D; Shin, Jung-A; Lim, Kayleen; Lee, Eun-Jin; Van Craenenbroeck, Kathleen; Craft, Cheryl Mae

2015-10-01

The G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors. To test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4. Immunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation. The results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization. Copyright © 2015 Elsevier Inc. All rights reserved.

The Increased Endogenous Sulfur Dioxide Acts as a Compensatory Mechanism for the Downregulated Endogenous Hydrogen Sulfide Pathway in the Endothelial Cell Inflammation

PubMed Central

Zhang, Da; Wang, Xiuli; Tian, Xiaoyu; Zhang, Lulu; Yang, Guosheng; Tao, Yinghong; Liang, Chen; Li, Kun; Yu, Xiaoqi; Tang, Xinjing; Tang, Chaoshu; Zhou, Jing; Kong, Wei; Du, Junbao; Huang, Yaqian; Jin, Hongfang

2018-01-01

Endogenous hydrogen sulfide (H2S) and sulfur dioxide (SO2) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H2S on the SO2 generation in the endothelial cells and explore its significance in the endothelial inflammation in vitro and in vivo. The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for in vitro experiments. A rat model of monocrotaline (MCT)-induced pulmonary vascular inflammation was used for in vivo experiments. We found that endogenous H2S deficiency caused by cystathionine-γ-lyase (CSE) knockdown increased endogenous SO2 level in endothelial cells and enhanced the enzymatic activity of AAT, a major SO2 synthesis enzyme, without affecting the expressions of AAT1 and AAT2. While H2S donor could reverse the CSE knockdown-induced increase in the endogenous SO2 level and AAT activity. Moreover, H2S donor directly inhibited the activity of purified AAT protein, which was reversed by a thiol reductant DTT. Mechanistically, H2S donor sulfhydrated the purified AAT1/2 protein and rescued the decrease in the sulfhydration of AAT1/2 protein in the CSE knockdown endothelial cells. Furthermore, an AAT inhibitor l-aspartate-β-hydroxamate (HDX), which blocked the upregulation of endogenous SO2/AAT generation induced by CSE knockdown, aggravated CSE knockdown-activated nuclear factor-κB pathway in the endothelial cells and its downstream inflammatory factors including ICAM-1, TNF-α, and IL-6. In in vivo experiment, H2S donor restored the deficiency of endogenous H2S production induced by MCT, and reversed the upregulation of endogenous SO2/AAT pathway via sulfhydrating AAT1 and AAT2. In accordance with the results of the in vitro experiment, HDX exacerbated the pulmonary vascular inflammation induced by the broken endogenous H2S production in MCT-treated rat. In conclusion, for the first time, the present study showed that H2S inhibited endogenous SO2 generation by inactivating AAT via the sulfhydration of AAT1/2; and the increased endogenous SO2 generation might play a compensatory role when H2S/CSE pathway was downregulated, thereby exerting protective effects in endothelial inflammatory responses in vitro and in vivo. PMID:29760703

Importance of perceived naturalness for acceptance of food additives and cultured meat.

PubMed

Siegrist, Michael; Sütterlin, Bernadette

2017-06-01

Four experiments examined some factors influencing the perceived naturalness of food products and their biasing effect on risk perception. The results of Experiment 1a showed that three food additives displaying their respective E-numbers (i.e., codes for food additives in the European Union and Switzerland) decreased perceived naturalness. Experiment 1b demonstrated that mentioning possible health effects decreased the perceived naturalness of a plant-based food additive. This experiment further showed that it would not matter for perceived naturalness whether the food was synthetic or nature-identical. Moreover, the results of Experiments 2 and 3 suggested that the same risk associated with meat consumption was much more acceptable for traditionally produced meat compared with in-vitro meat. Experiment 3 further indicated that the perceived naturalness of the meat (i.e., traditional or cultured meat) had a full mediation effect on participants' evaluation of the acceptability of the risk of colon cancer associated with the meat consumption. Even if the new production method (i.e., cultured meat) was more environmentally friendly and less harmful to animals, the perceived lack of naturalness might reduce the acceptability of the risk associated with such a product. The present study provides evidence that consumers rely on symbolic information when evaluating foods, which may lead to biased judgments and decisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural Iron (II) of Basaltic Glass as an Energy Source for Zetaproteobacteria in an Abyssal Plain Environment, Off the Mid Atlantic Ridge

PubMed Central

Henri, Pauline A.; Rommevaux-Jestin, Céline; Lesongeur, Françoise; Mumford, Adam; Emerson, David; Godfroy, Anne; Ménez, Bénédicte

2016-01-01

To explore the capability of basaltic glass to support the growth of chemosynthetic microorganisms, complementary in situ and in vitro colonization experiments were performed. Microbial colonizers containing synthetic tholeitic basaltic glasses, either enriched in reduced or oxidized iron, were deployed off-axis from the Mid Atlantic Ridge on surface sediments of the abyssal plain (35°N; 29°W). In situ microbial colonization was assessed by sequencing of the 16S rRNA gene and basaltic glass alteration was characterized using Scanning Electron Microscopy, micro-X-ray Absorption Near Edge Structure at the Fe-K-edge and Raman microspectroscopy. The colonized surface of the reduced basaltic glass was covered by a rind of alteration made of iron-oxides trapped in a palagonite-like structure with thicknesses up to 150 μm. The relative abundance of the associated microbial community was dominated (39% of all reads) by a single operational taxonomic unit (OTU) that shared 92% identity with the iron-oxidizer Mariprofundus ferrooxydans PV-1. Conversely, the oxidized basaltic glass showed the absence of iron-oxides enriched surface deposits and correspondingly there was a lack of known iron-oxidizing bacteria in the inventoried diversity. In vitro, a similar reduced basaltic glass was incubated in artificial seawater with a pure culture of the iron-oxidizing M. ferrooxydans DIS-1 for 2 weeks, without any additional nutrients or minerals. Confocal Laser Scanning Microscopy revealed that the glass surface was covered by twisted stalks characteristic of this iron-oxidizing Zetaproteobacteria. This result supported findings of the in situ experiments indicating that the Fe(II) present in the basalt was the energy source for the growth of representatives of Zetaproteobacteria in both the abyssal plain and the in vitro experiment. In accordance, the surface alteration rind observed on the reduced basaltic glass incubated in situ could at least partly result from their activity. PMID:26834704

[Anti-tumor effect of 5-FU-PLLA-CNTs on human gastric carcinoma cell lines in vitro].

PubMed

Gu, Jun; Li, Maolan; Wu, Xiangsong; Wu, Wenguang; Zhang, Lin; Ding, Qichen; Yang, Jiahua; Weng, Hao; Ding, Qian; Bao, Runfa; Shu, Yijun; Liu, Yingbin

2014-04-01

To prepare cisPLLAtin-loaded polylactic acid/cnts, and to study the anti-tumor effect of 5-FU-PLLA-CNTs on human gastric carcinoma cell lines(MGC803 and MNK45). 5-FU-PLLA-CNTs were prepared with ultrasound emulsification. The morphology of 5-FU-PLLA-CNTs was determined by scanning electron microscope(SEM), and its drug loading and drug release curve in vitro were detected by UV-Vis-NIR spectrophotometer. Cells were divided into experiment, positive control and negative control groups. CCK8 method was used to test the cytotoxic effect of 5-FU-PLLA-CNTs in different concentrations on MGC803 and MNK45 cell proliferation. Flow cytometry was employed to measure the apoptotic rate of MGC803 and MNK45 cells before and after the intervention of 5-FU-PLLA-CNTs. Deep layer film of 5-FU-PLLA-CNTs was successfully established, whose drug-load rate was(4.54±0.43)%, entrapment rate was(21.56±2.36)%. In vitro release test showed release rate within 24 h of 5-FU-PLLA-CNTs was 23.9% in a as lowly increasing manner, and accumulating release rate was 85.3% at day 31. CCk8 experiment revealed, as compared to control group, 5-FU-PLLA-CNTs significantly inhibited the proliferation of two cell lines in dose-dependent and time-dependent manner. The best 5-FU-PLLA-CNTs concentration of inhibition for human gastric cancer cell lines was 1 mg/well. Flow cytometry indicated the apoptotic rate of MGC803 and MNK45 cells in experiment group treated by 1 mg/well 5-FU-PLLA-CNTs significantly increased as compared to negative control group (P<0.05), while the difference was not significant as compared to positive control group (P>0.05). The 5-FU-PLLA-CNTs has good drug sustained-release capacity, and can significantly kill and inhibit the proliferation of MGC803 and MNK45 cell lines.

Review: Oxygen and trophoblast biology — A source of controversy

PubMed Central

Tuuli, M.G.; Longtine, M.S.; Nelson, D.M.

2013-01-01

Oxygen is necessary for life yet too much or too little oxygen is toxic to cells. The oxygen tension in the maternal plasma bathing placental villi is <20 mm Hg until 10–12 weeks’ gestation, rising to 40–80 mmHg and remaining in this range throughout the second and third trimesters. Maldevelopment of the maternal spiral arteries in the first trimester predisposes to placental dysfunction and sub-optimal pregnancy outcomes in the second half of pregnancy. Although low oxygen at the site of early placental development is the norm, controversy is intense when investigators interpret how defective transformation of spiral arteries leads to placental dysfunction during the second and third trimesters. Moreover, debate rages as to what oxygen concentrations should be considered normal and abnormal for use in vitro to model villous responses in vivo. The placenta may be injured in the second half of pregnancy by hypoxia, but recent evidence shows that ischemia with reoxygenation and mechanical damage due to high flow contributes to the placental dysfunction of diverse pregnancy disorders. We overview normal and pathologic development of the placenta, consider variables that influence experiments in vitro, and discuss the hotly debated question of what in vitro oxygen percentage reflects the normal and abnormal oxygen concentrations that occur in vivo. We then describe our studies that show cultured villous trophoblasts undergo apoptosis and autophagy with phenotype-related differences in response to hypoxia. PMID:21216006

Preparation and in vitro characterization of chitosan nanobubbles as theranostic agents.

PubMed

Cavalli, R; Argenziano, M; Vigna, E; Giustetto, P; Torres, E; Aime, S; Terreno, E

2015-05-01

Theranostic delivery systems are nanostructures that combine the modality of therapy and diagnostic imaging. Polymeric micro- and nanobubbles, spherical vesicles containing a gas core, have been proposed as new theranostic carriers for MRI-guided therapy. In this study, chitosan nanobubbles were purposely tuned for the co-delivery of prednisolone phosphate and a Gd(III) complex, as therapeutic and MRI diagnostic agent, respectively. Perfluoropentane was used for filling up the internal core of the formulation. These theranostic nanobubbles showed diameters of about 500nm and a positive surface charge that allows the interaction with the negatively charged Gd-DOTP complex. Pluronic F68 was added to the nanobubble aqueous suspension as stabilizer agent. The encapsulation efficiency was good for both the active compounds, and a prolonged drug release profile was observed in vitro. The effect of ultrasound stimulation on prednisolone phosphate release was evaluated at 37°C. A marked increase on drug release kinetics with no burst effect was obtained after the exposure of the system to ultrasound. Furthermore, the relaxivity of the MRI probe changed upon incorporation in the nanobubble shell, thereby offering interesting opportunity in dual MRI-US experiments. The ultrasound characterization showed a good in vitro echogenicity of the theranostic nanobubbles. In summary, chitosan drug-loaded nanobubbles with Gd(III) complex bound to their shell might be considered a new platform for imaging and drug delivery with the potential of improving anti-cancer treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

In vitro and in vivo antiviral properties of sulfated galactomannans against yellow fever virus (BeH111 strain) and dengue 1 virus (Hawaii strain).

PubMed

Ono, Lucy; Wollinger, Wagner; Rocco, Iray M; Coimbra, Terezinha L M; Gorin, Philip A J; Sierakowski, Maria-Rita

2003-11-01

Two galactomannans, one extracted from seeds of Mimosa scabrella, having a mannose to galactose ratio of 1.1, and another with a 1.4 ratio from seeds of Leucaena leucocephala, were sulfated. The products from M. scabrella (BRS) and L. leucocephala (LLS) had a degree of sulfation of 0.62 and 0.50, and an average molecular weight of 620x10(3) and 574x10(3) gmol(-1), respectively. Their activities against yellow fever virus (YFV; BeH111 strain) and dengue 1 virus (DEN-1; Hawaii strain) were evaluated. This was carried out in young mice following intraperitoneal infection with YFV. At a dose of 49 mgkg(-1), BRS and LLS gave protection against death in 87.7 and 96.5% of the mice, respectively. When challenged with 37.5 LD50 of YFV, mice previously inoculated with BRS+virus or LLS+virus, showed 93.3 and 100% resistance, respectively, with neutralization titers similar to mice injected with 25 LD50 of formaldehyde-inactivated YFV. In vitro experiments with YFV and DEN-1 in C6/36 cell culture assays in 24-well microplates showed that concentrations that produced a 100-fold decrease in virus titer of YFV were 586 and 385 mgl(-1) for BRS and LLS, respectively. For DEN-1 they were 347 and 37 mgl(-1), respectively. Sulfated galactomannans, thus demonstrate in vitro and in vivo activity against flaviviruses.

Antituberculous effect of silver nanoparticles

NASA Astrophysics Data System (ADS)

Kreytsberg, G. N.; Gracheva, I. E.; Kibrik, B. S.; Golikov, I. V.

2011-04-01

The in vitro experiment, involving 1164 strains of the tuberculosis mycobacteria, exhibited a potentiating effect of silver nanoparticles on known antituberculous preparations in respect of overcoming drug-resistance of the causative agent. The in vitro experiment, based on the model of resistant tuberculosis, was performed on 65 white mice. An evident antituberculous effect of the nanocomposite on the basis of silver nanoparticles and isoniazid was proved. Toxicological assessment of the of nanopreparations was carried out. The performed research scientifically establishes efficacy and safety of the nanocomposite application in combination therapy of patients suffering from drug-resistant tuberculosis.

Hydroxycinnamate conjugates as potential monolignol replacements: In vitro lignification and cell wall studies with rosmarinic acid

USDA-ARS?s Scientific Manuscript database

The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers such as rosmarinic acid (RA) and analogous catechol derivatives to create cell wall lignins that are less recalcitrant to biomass processing. In vitro lignin polymerization experiments revealed that...

International standardization of cage designs and feeding regimes for honey bee in vitro experiments

USDA-ARS?s Scientific Manuscript database

The aim of this study was to improve and standardize cage systems for maintaining adult honey bee workers under in vitro laboratory conditions. To achieve this goal, we experimentally evaluated the impact of different cages, developed by scientists of the international research network COLOSS (Preve...

Basic fibroblastic growth factor affects the osteogenic differentiation of dental pulp stem cells in a treatment-dependent manner.

PubMed

Qian, J; Jiayuan, W; Wenkai, J; Peina, W; Ansheng, Z; Shukai, S; Shafei, Z; Jun, L; Longxing, N

2015-07-01

To determine how basic fibroblastic growth factor (bFGF) affected the osteogenic differentiation of human dental pulp stem cells (DPSCs) in vitro and in vivo. Basic fibroblastic growth factor stimulation of DPSCs was divided into a pre-treatment period and an osteogenic differentiation period. Alizarin red quantification experiments and alkaline phosphatase activity quantification assay were performed to examine the osteogenic differentiation of DPSCs after different bFGF stimulation. Quantification reverse transcription polymerase chain reaction was used to analyze the osteogenic gene expression of DPSCs after different bFGF stimulation. In addition, DPSCs that received the 1 and 2 weeks bFGF pre-treatments as in the in vitro experiments were mineralized for 1 week and seeded into hydroxyapatite/tricalcium phosphate (HA/TCP) pills and subcutaneously transplanted into naked mice for 2 or 3 months. The transplants were removed, sliced and stained using Modified Ponceau Trichrome Stain to observe the formation of mineralized tissue. Basic fibroblastic growth factor stimulation in the osteogenic differentiation period decreased the in vitro osteogenic differentiation ability of DPSCs. One week pre-treatment with bFGF increased the in vitro osteogenic differentiation ability of DPSCs, whereas 2 weeks pre-treatment with bFGF decreased the in vitro osteogenic differentiation ability of DPSCs. The pre-treatment period was vital for the osteogenic differentiation of DPSCs in vitro. The in vivo results were similar to the in vitro results. Basic fibroblastic growth factor affected the osteogenic differentiation of DPSCs in a treatment-dependent manner both in vitro and in vivo. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Isolation of Antagonistic Endophytes from Banana Roots against Meloidogyne javanica and Their Effects on Soil Nematode Community

PubMed Central

Su, Lanxi; Shen, Zongzhuan; Ruan, Yunze; Tao, Chengyuan; Chao, Yifan; Li, Rong; Shen, Qirong

2017-01-01

Banana production is seriously hindered by Meloidogyne spp. all over the world. Endophytes are ideal candidates compared to pesticides as an environmentally benign agent. In the present study, endophytes isolated from banana roots infected by Meloidogyne spp. with different disease levels were tested in vitro, and in sterile and nature banana monoculture soils against Meloidogyne javanica. The proportion of antagonistic endophytes were higher in the roots of middle and high disease levels. Among those, bacteria were dominant, and Pseudomonas spp., Bacillus spp. and Streptomyces spp. showed more abundant populations. One strain, named as SA, with definite root inner-colonization ability was isolated and identified as Streptomyces sp. This strain showed an inhibiting rate of >50% in vitro and biocontrol efficiency of 70.7% in sterile soil against Meloidogyne javanica, compared to the control. Greenhouse experiment results showed that the strain SA exhibits excellent biological control ability for plant-parasites both in roots and in root-knot nematode infested soil. SA treatment showed a higher number of bacterivores, especially Mesorhabditis and Cephalobus. The maturity index was significantly lower, while enrichment index (EI) was significantly higher in the SA treatment. In conclusion, this study presents an important potential application of the endophytic strain Streptomyces sp. for the control of plant-parasitic nematodes, especially Meloidogyne javanica, and presents the effects on the associated variation of the nematode community. PMID:29123509

[Studies on the factors responsible for low erythrocyte alpha-tocopherol concentrations in patients on maintenance hemodialysis].

PubMed

Mori, K

1989-03-01

The present study was undertaken to clarify the low erythrocyte (RBC) alpha-tocopherol (TOC) concentrations in patients on maintenance hemodialysis (HD) using in vivo and in vitro experiments. 15 age-matched male controls and HD patients were evaluated. The experimental designs and the results were as follows: 1) Changes in fasting stage; TOC levels of plasma, high density lipoproteins (HDL) and RBC, especially RBC, were lower in HD-patients than in controls. The ratio of RBC to plasma and HDL showed more significant changes between HD-patients and controls. In controls, RBC-TOC was noted to have a positive correlation with HDL-TOC and a negative correlation with non HDL-(plasma--HDL)-TOC, whereas a reverse correlation was observed in HD-patients. 2) In vivo experiments: Each 7 of HD-patients and controls were orally given TOC 600 mg after meal at early morning; the blood was sampled at 0, 3, 6, 10 and 24 hr. Poor increase of RBC-TOC in HD-patients was observed. In contrast, HDL-TOC was significantly higher at all measurement times than in controls. The change of plasma-TOC was similar to that of non HDL-TOC. 3) In vitro experiment:RBC from patients or controls showing various concentrations of RBC-TOC and plasma from controls were incubated at 37 degrees C or 4 degrees C. The inactivated plasma from controls was prepared by incubation at 56 degrees C, 30 min or by supplement of 2 mM parachloromerucuric benzonic sulfonate. The ratio of RBC and plasma was at 2:3 close to physiological conditions. No difference between patient and control RBCs as an acceptor of TOC and no appreciable transfer of TOC between RBC to plasma at 4 degrees C were observed. When RBC-TOC was low, transfer volume of TOC from plasma to RBC was increased and TOC was supplied from mainly non HDL fractions; in contrast, when it was high, the main source of TOC was due to the transfer from HDL fractions which was reduced when lecithin: cholesterol acyltransferase (LCAT) reaction was inhibited. From above in vivo and in vitro studies, it was concluded that low levels of RBC-TOC in HD-patients may be due to an insufficiency of TOC transfer from HDL fractions affected by low LCAT activity.

A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

PubMed

Nogami, Makiko; Kimura, Tomoatsu; Seki, Shoji; Matsui, Yoshito; Yoshida, Toshiko; Koike-Soko, Chika; Okabe, Motonori; Motomura, Hiraku; Gejo, Ryuichi; Nikaido, Toshio

2016-04-01

Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery.

PubMed

Yao, Yao; Su, Zhihui; Liang, Yanchao; Zhang, Na

2015-01-01

Combination of chemotherapeutic drug and small interfering RNA (siRNA) can affect multiple disease pathways and has been proven effective in suppressing tumor progression. Co-delivery of drug and siRNA within a same nanocarrier is a vital means in this field. The present study aimed at the development of a pH-sensitive liposome to co-deliver drug and siRNA to tumor region. Driven by the electrostatic interaction, the pH-sensitive material, carboxymethyl chitosan (CMCS), was coated onto the surface of the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si). To evaluate whether the resulting CMCS-modified Sf and siRNA co-delivery cationic liposome (CMCS-SiSf-CL) enhanced antitumor efficiency after systematic administration, in vitro and in vivo experiments were evaluated in HepG2 cells and the H22 cells-bearing Kunming mice model. The experimental results demonstrated that CMCS-SiSf-CL was able to condense siRNA efficiently and protect siRNA from being degraded by serum and RNase. The release rate of Sf from CMCS-modified liposome exhibited pH-sensitive release behavior. Furthermore, in vitro cellular uptake results showed that CMCS-SiSf-CL yielded higher fluorescence intensity at pH 6.5 than at pH 7.4, and that siRNA could be delivered to tumor site by CMCS-SiSf-CL in vivo. The in vivo antitumor efficacy showed that CMCS-Sf-CL inhibits tumor growth effectively when compared with free Sf solution. In current experimental conditions, this liposomal formulation did not show significant toxicity both in vitro and in vivo. Therefore, co-delivering Sf with siRNA by CMCS-SiSf-CL might provide a promising approach for tumor therapy.

In vitro and in vivo corrosion properties of new iron-manganese alloys designed for cardiovascular applications.

PubMed

Drynda, Andreas; Hassel, Thomas; Bach, Friedrich Wilhelm; Peuster, Matthias

2015-04-01

The principle of biodegradation for the production of temporary implant materials (e.g. stents) plays an important role in the treatment of congenital heart defects. In the last decade several attempts have been made with different alloy materials-mainly based on iron and magnesium. None of the currently available materials in this field have demonstrated satisfying results and have therefore not found entry into broad clinical practice. While magnesium or magnesium alloy systems corrode too fast, the corrosion rate of pure iron-stents is too slow for cardiovascular applications. In the last years FeMn alloy systems were developed with the idea that galvanic effects, caused by different electrochemical properties of Fe and Mn, would increase the corrosion rate. In vitro tests with alloys containing up to 30% Mn showed promising results in terms of biocompatibility. This study deals with the development of new FeMn alloy systems with lower Mn concentrations (FeMn 0.5 wt %, FeMn 2.7 wt %, FeMn 6.9 wt %) to avoid Mn toxicity. Our results show, that these alloys exhibit good mechanical features as well as suitable in vitro biocompatibility and corrosion properties. In contrast, the evaluation of these alloys in a mouse model led to unexpected results-even after 9 months no significant corrosion was detectable. Preliminary SEM investigations showed that passivation layers (FeMn phosphates) might be the reason for corrosion resistance. If this can be proved in further experiments, strategies to prevent or dissolve those layers need to be developed to expedite the in vivo corrosion of FeMn alloys. © 2014 Wiley Periodicals, Inc.

Sparse matrix beamforming and image reconstruction for 2-D HIFU monitoring using harmonic motion imaging for focused ultrasound (HMIFU) with in vitro validation.

PubMed

Hou, Gary Y; Provost, Jean; Grondin, Julien; Wang, Shutao; Marquet, Fabrice; Bunting, Ethan; Konofagou, Elisa E

2014-11-01

Harmonic motion imaging for focused ultrasound (HMIFU) utilizes an amplitude-modulated HIFU beam to induce a localized focal oscillatory motion simultaneously estimated. The objective of this study is to develop and show the feasibility of a novel fast beamforming algorithm for image reconstruction using GPU-based sparse-matrix operation with real-time feedback. In this study, the algorithm was implemented onto a fully integrated, clinically relevant HMIFU system. A single divergent transmit beam was used while fast beamforming was implemented using a GPU-based delay-and-sum method and a sparse-matrix operation. Axial HMI displacements were then estimated from the RF signals using a 1-D normalized cross-correlation method and streamed to a graphic user interface with frame rates up to 15 Hz, a 100-fold increase compared to conventional CPU-based processing. The real-time feedback rate does not require interrupting the HIFU treatment. Results in phantom experiments showed reproducible HMI images and monitoring of 22 in vitro HIFU treatments using the new 2-D system demonstrated reproducible displacement imaging, and monitoring of 22 in vitro HIFU treatments using the new 2-D system showed a consistent average focal displacement decrease of 46.7 ±14.6% during lesion formation. Complementary focal temperature monitoring also indicated an average rate of displacement increase and decrease with focal temperature at 0.84±1.15%/(°)C, and 2.03±0.93%/(°)C , respectively. These results reinforce the HMIFU capability of estimating and monitoring stiffness related changes in real time. Current ongoing studies include clinical translation of the presented system for monitoring of HIFU treatment for breast and pancreatic tumor applications.

Effects of Nicotine Exposure on In Vitro Metabolism of Chlorpyrifos in Male Sprague-Dawley Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sookwang; Busby, Andrea L.; Timchalk, Charles

Chlorpyrifos (CPF) is a common organophosphate (OP) insecticide which is metabolized by CYP450s to the neurotoxic metabolite, chlorpyrifos-oxon (CPF-oxon) and a non-toxic metabolite, 3,5,6-trichloro-2-pyridinol (TCP). The objective of this study was to quantify the effect of repeated in vivo nicotine exposures on CPF in vitro metabolism and marker substrate activities in rats. Male Sprague-Dawley rats were dosed subcutaneously with 1 mg nicotine/kg/, for up to 10 days. Animals showed signs of cholinergic crisis after the initial nicotine doses, but exhibited adaptation after a couple days of treatment. Rats were sacrificed on selected days 4 or 24 hr after the lastmore » nicotine-treatment. While CYP450 reduced CO spectra were not different across the treatments, the single nicotine dose group showed a 2-fold increase in CYP2E1 marker substrate (p-nitrophenol) activity 24 hr after a single nicotine treatment compared to saline controls. Conversely, repeated nicotine treatments resulted in decreased EROD marker substrate activity 4 hr after the 7th day of treatment. CPF-oxon Vmax and Km did not show significant changes across the different nicotine treatment groups. The Vmax describing the metabolism of CPF to TCP was increased on all groups (days 1, 7, and 10) 24 hr after nicotine treatment but were unchanged 4 hr after nicotine treatment. Results of this in vitro study suggest that repeated nicotine exposure (i.e., from smoking) may result in altered metabolism of CPF. Future in vivo experiments based on these results will be conducted to ascertain the impact of in vivo nicotine exposures on CPF metabolism in rats.« less

In-vitro evaluation of Polylactic acid (PLA) manufactured by fused deposition modeling.

PubMed

Wurm, Matthias C; Möst, Tobias; Bergauer, Bastian; Rietzel, Dominik; Neukam, Friedrich Wilhelm; Cifuentes, Sandra C; Wilmowsky, Cornelius von

2017-01-01

With additive manufacturing (AM) individual and biocompatible implants can be generated by using suitable materials. The aim of this study was to investigate the biological effects of polylactic acid (PLA) manufactured by Fused Deposition Modeling (FDM) on osteoblasts in vitro according to European Norm / International Organization for Standardization 10,993-5. Human osteoblasts (hFOB 1.19) were seeded onto PLA samples produced by FDM and investigated for cell viability by fluorescence staining after 24 h. Cell proliferation was measured after 1, 3, 7 and 10 days by cell-counting and cell morphology was evaluated by scanning electron microscopy. For control, we used titanium samples and polystyrene (PS). Cell viability showed higher viability on PLA (95,3% ± 2.1%) than in control (91,7% ±2,7%). Cell proliferation was highest in the control group (polystyrene) and higher on PLA samples compared to the titanium samples. Scanning electron microscopy revealed homogenous covering of sample surface with regularly spread cells on PLA as well as on titanium. The manufacturing of PLA discs from polylactic acid using FDM was successful. The in vitro investigation with human fetal osteoblasts showed no cytotoxic effects. Furthermore, FDM does not seem to alter biocompatibility of PLA. Nonetheless osteoblasts showed reduced growth on PLA compared to the polystyrene control within the cell experiments. This could be attributed to surface roughness and possible release of residual monomers. Those influences could be investigated in further studies and thus lead to improvement in the additive manufacturing process. In addition, further research focused on the effect of PLA on bone growth should follow. In summary, PLA processed in Fused Deposition Modelling seems to be an attractive material and method for reconstructive surgery because of their biocompatibility and the possibility to produce individually shaped scaffolds.

The effect of microgravity on the in vitro NK cell function during six International Space Station Missions

NASA Astrophysics Data System (ADS)

Buravkova, L. B.; Grigorieva, V.; Rykova, M. P.

2007-09-01

The level of natural killer (NK) cytotoxic activity was measured during co-cultivation of human lymphocytes and target cells (K-562) in microgravity. Flight experiments were carried out using special instrumentation, the "Fibroblast-1 " cassettes, in the frame of Russian scientific program during six ISS missions. Lymphocyte suspensions from human venous blood were used in experiments during short-term flights on six ISS missions (7-12). Russian space crew members performed the experiments after Soyuz docking. The first step was mixing lymphocytes and3H-labeled K-562 cells and their incubation at 37°C during 24 hs; the second step was filtration of the cell suspension. The frozen medium and filters were analyzed for the cytokine level and cytotoxic activity after landing. It was found that lymphocytes with different basal levels of cytotoxic activity kept the ability of recognizing and lysing malignant cells. In microgravity, cytotoxity increased to 160% of the basal levels. Donor individual features modulated the magnitude of the increase. The measurement of interleukin levels (TNF-α, IL-1, IL-2) in medium showed that synthesis of TNF-α increased during cell co-cultivation in microgravity. The level of IL-2 was very low inflight and ground control samples. The production of IL-1 by lymphocytes decreased after in-flight incubation. The results indicate that microgravity did not disturb the cytotoxic function of immune cells in vitro during 24 h incubation with specific target cells.

Prior reproductive experience alters prolactin-induced macrophage responses in pregnant rats.

PubMed

Carvalho-Freitas, Maria Isabel Roth; Anselmo-Franci, Janete A; Palermo-Neto, João; Felicio, Luciano F

2013-09-01

Reproductive experience (i.e., pregnancy and lactation) induces physiological changes in mammals. A previous reproductive experience was recently shown to modulate the activity of dopaminergic hypothalamic systems while decreasing serum prolactin levels and oxidative burst activity in peritoneal macrophages. Dopamine receptor antagonists increase serum prolactin levels, and both prolactin and dopamine receptors may be involved in the modulation of macrophage activity, providing a means of communication between the nervous and immune systems. The present study evaluated the in vitro effects of prolactin and a dopamine D2 receptor antagonist on the peritoneal activity of macrophages from primigravid and multigravid female rats during the third trimester of pregnancy. Oxidative bursts and phagocytosis in peritoneal macrophages were evaluated by flow cytometry. Primigravid and multigravid Wistar rats, during the third trimester of pregnancy (i.e., days 17-21), were used. Peritoneal fluid samples from these rats were first incubated with prolactin (10 and 100 nM) for different periods of time. The same procedure was repeated to evaluate the effects of domperidone (10 and 100 nM) on macrophage activity. Our results showed that macrophages from multigravid rats responded more effectively to in vitro incubation with prolactin, especially with regard to the intensity and percentage of phagocytosis. Additionally, these effects were more pronounced after incubation periods of 30 min or 4 h. These data suggest that macrophages during a second pregnancy become more sensitive to the phagocytotic effects of prolactin. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Impact of transcytolemmal water exchange on estimates of tissue microstructural properties derived from diffusion MRI.

PubMed

Li, Hua; Jiang, Xiaoyu; Xie, Jingping; Gore, John C; Xu, Junzhong

2017-06-01

To investigate the influence of transcytolemmal water exchange on estimates of tissue microstructural parameters derived from diffusion MRI using conventional PGSE and IMPULSED methods. Computer simulations were performed to incorporate a broad range of intracellular water life times τ in (50-∞ ms), cell diameters d (5-15 μm), and intrinsic diffusion coefficient D in (0.6-2 μm 2 /ms) for different values of signal-to-noise ratio (SNR) (10 to 50). For experiments, murine erythroleukemia (MEL) cancer cells were cultured and treated with saponin to selectively change cell membrane permeability. All fitted microstructural parameters from simulations and experiments in vitro were compared with ground-truth values. Simulations showed that, for both PGSE and IMPULSED methods, cell diameter d can be reliably fit with sufficient SNR (≥ 50), whereas intracellular volume fraction f in is intrinsically underestimated due to transcytolemmal water exchange. D in can be reliably fit only with sufficient SNR and using the IMPULSED method with short diffusion times. These results were confirmed with those obtained in the cell culture experiments in vitro. For the sequences and models considered in this study, transcytolemmal water exchange has minor effects on the fittings of d and D in with physiologically relevant membrane permeabilities if the SNR is sufficient (> 50), but f in is intrinsically underestimated. Magn Reson Med 77:2239-2249, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro.

PubMed

Roasa, L M; Choi, Y H; Love, C C; Romo, S; Varner, D D; Hinrichs, K

2007-09-01

In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates were influenced by factors associated with the freezing process (Experiment 2), a single ejaculate from a second stallion was frozen using eight variations in timing of steps in the freezing protocol. There were no differences among treatments in fertilization rates (range, 0-3%). In Experiment 3, fertilization rates of semen frozen in an extender containing 21.5% egg yolk were lower than fertilization rates of semen from the same ejaculate but frozen with a 3% egg-yolk extender (0% versus 15%, respectively; P<0.01). We inferred that rates of equine IVF with frozen-thawed sperm were influenced by ejaculate, the composition and age of the media used, and freezing extender. To our knowledge, this is the first report of ejaculate or extender differences affecting in vitro fertilization in this species. These factors may help to explain the great variability in fertilization rates reported with equine IVF, both among and within laboratories.

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

PubMed Central

Struffi, Paolo; Corado, Maria; Kaplan, Leah; Yu, Danyang; Rushlow, Christine; Small, Stephen

2011-01-01

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo. PMID:21865322

In vitro-in vivo study on the effects of plant compounds on rumen fermentation, microbial abundances and methane emissions in goats.

PubMed

Martínez-Fernández, G; Abecia, L; Martín-García, A I; Ramos-Morales, E; Hervás, G; Molina-Alcaide, E; Yáñez-Ruiz, D R

2013-12-01

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P≥ 0.366). Total volatile fatty-acid (VFA) concentration decreased (P≤ 0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P≤ 0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P≤ 0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P≤ 0.004) by CAR and PTS. The addition of all compounds linearly decreased (P≤ 0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet-dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P≤ 0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.

In vitro study on effect of germinated wheat on human breast cancer cells

USDA-ARS?s Scientific Manuscript database

This research investigated the possible anti-cancer effects of germinated wheat flours (GWF) on cell growth and apoptosis of human breast cancer cells. In a series of in vitro experiments, estrogen receptor-positive (MCF-7) and negative (MDA-MB-231) cells were cultured and treated with GWF that wer...

Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

COMPARISON OF IN VIVO DERIVED AND SCALED IN VITRO METABOLIC RATE CONSTANTS FOR SOME VOLATILE ORGANIC COMPOUNDS (VOCS)

EPA Science Inventory

The reliability of physiologically based pharmacokinetic (PBPK) models is directly related to the accuracy of the metabolic rate parameters used as model inputs. When metabolic rate parameters derived from in vivo experiments are unavailable, they can be estimated from in vitro d...

Formulation of diazepam containing rectal suppositories and experiences of their biopharmaceutical study.

PubMed

Regdon, G; Bácskay, I; Kata, M; Selmeczi, B; Szikszay, M; Sánta, A; Bálint, G S

1994-05-01

Methodology and the results of the in vitro membrane diffusion and in vivo bioavailability studies are presented. The results confirm a correlation between in vitro and in vivo findings. Hydrophilic macrogol-mixture with great molecular mass can be recommended as the optimal vehicle for formulation of diazepam suppositories.

Toxic effects of ethylene oxide residues on bovine embryos in vitro.

PubMed

Holyoak, G R; Wang, S; Liu, Y; Bunch, T D

1996-04-15

The potential of ethylene oxide (EtO) residues in exposed plastic tissue culture dishes to adversely affect bovine oocyte maturation, fertilization and subsequent embryonic development was monitored. In experiment 1, the effects of aeration time and aeration combined with washing of EtO-gassed culture dishes on the extent of residual toxicity were investigated. There was no cleavage in any treatment in which oocytes were matured and fertilized in dishes exposed to EtO. EtO residues caused functional degeneration of oocytes even when culture dishes were aerated for more than 12 days post EtO-exposure and repeatedly washed. In experiment 2, the residual toxicity of EtO gas on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were evaluated. Cleavage rate significantly decreased and post-cleavage development was retarded in ova maintained in dishes treated with EtO either during IVM or IVF. EtO residues may be more detrimental to spermatozoa than to oocytes which may have been the primary cause of fertilization failure during IVF.

Codelivery of doxorubicin and triptolide with reduction-sensitive lipid–polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment

PubMed Central

Wu, Bo; Lu, Shu-Ting; Zhang, Liu-Jie; Zhuo, Ren-Xi; Xu, Hai-Bo; Huang, Shi-Wen

2017-01-01

Codelivery is a promising strategy to overcome the limitations of single chemotherapeutic agents in cancer treatment. Despite progress, codelivery of two or more different functional drugs to increase anticancer efficiency still remains a challenge. Here, reduction-sensitive lipid–polymer hybrid nanoparticles (LPNPs) drug delivery system composed of monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16), soybean lecithin, and poly(D,L-lactide-co-glycolide) (PLGA) was used for codelivery of doxorubicin (DOX) and a Chinese herb extract triptolide (TPL). Hydrophobic DOX and TPL could be successfully loaded in LPNPs by self-assembly. More importantly, drug release and cellular uptake experiments demonstrated that the two drugs were reduction sensitive, released simultaneously from LPNPs, and taken up effectively by the tumor cells. DOX/TPL-coloaded LPNPs (DOX/TPL-LPNPs) exhibited a high level of synergistic activation with low combination index (CI) in vitro and in vivo. Moreover, the highest synergistic therapeutic effect was achieved at the ratio of 1:0.2 DOX/TPL. Further experiments showed that TPL enhanced the uptake of DOX by human oral cavity squamous cell carcinoma cells (KB cells). Overall, DOX/TPL-coencapsulated reduction-sensitive nanoparticles will be a promising strategy for cancer treatment. PMID:28331310

Codelivery of doxorubicin and triptolide with reduction-sensitive lipid-polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment.

PubMed

Wu, Bo; Lu, Shu-Ting; Zhang, Liu-Jie; Zhuo, Ren-Xi; Xu, Hai-Bo; Huang, Shi-Wen

2017-01-01

Codelivery is a promising strategy to overcome the limitations of single chemotherapeutic agents in cancer treatment. Despite progress, codelivery of two or more different functional drugs to increase anticancer efficiency still remains a challenge. Here, reduction-sensitive lipid-polymer hybrid nanoparticles (LPNPs) drug delivery system composed of monomethoxy-poly(ethylene glycol)- S - S -hexadecyl (mPEG- S - S -C 16 ), soybean lecithin, and poly(D,L-lactide-co-glycolide) (PLGA) was used for codelivery of doxorubicin (DOX) and a Chinese herb extract triptolide (TPL). Hydrophobic DOX and TPL could be successfully loaded in LPNPs by self-assembly. More importantly, drug release and cellular uptake experiments demonstrated that the two drugs were reduction sensitive, released simultaneously from LPNPs, and taken up effectively by the tumor cells. DOX/TPL-coloaded LPNPs (DOX/TPL-LPNPs) exhibited a high level of synergistic activation with low combination index (CI) in vitro and in vivo. Moreover, the highest synergistic therapeutic effect was achieved at the ratio of 1:0.2 DOX/TPL. Further experiments showed that TPL enhanced the uptake of DOX by human oral cavity squamous cell carcinoma cells (KB cells). Overall, DOX/TPL-coencapsulated reduction-sensitive nanoparticles will be a promising strategy for cancer treatment.

On the Determination of Magnesium Degradation Rates under Physiological Conditions

PubMed Central

Nidadavolu, Eshwara Phani Shubhakar; Feyerabend, Frank; Ebel, Thomas; Willumeit-Römer, Regine; Dahms, Michael

2016-01-01

The current physiological in vitro tests of Mg degradation follow the procedure stated according to the ASTM standard. This standard, although useful in predicting the initial degradation behavior of an alloy, has its limitations in interpreting the same for longer periods of immersion in cell culture media. This is an important consequence as the alloy’s degradation is time dependent. Even if two different alloys show similar corrosion rates in a short term experiment, their degradation characteristics might differ with increased immersion times. Furthermore, studies concerning Mg corrosion extrapolate the corrosion rate from a single time point measurement to the order of a year (mm/y), which might not be appropriate because of time dependent degradation behavior. In this work, the above issues are addressed and a new methodology of performing long-term immersion tests in determining the degradation rates of Mg alloys was put forth. For this purpose, cast and extruded Mg-2Ag and powder pressed and sintered Mg-0.3Ca alloy systems were chosen. DMEM Glutamax +10% FBS (Fetal Bovine Serum) +1% Penicillin streptomycin was used as cell culture medium. The advantages of such a method in predicting the degradation rates in vivo deduced from in vitro experiments are discussed. PMID:28773749

Effect of ultra-high pressure homogenization on the interaction between bovine casein micelles and ritonavir.

PubMed

Corzo-Martínez, M; Mohan, M; Dunlap, J; Harte, F

2015-03-01

The aim of this work was to develop a milk-based powder formulation appropriate for pediatric delivery of ritonavir (RIT). Ultra-high pressure homogenization (UHPH) at 0.1, 300 and 500 MPa was used to process a dispersion of pasteurized skim milk (SM) and ritonavir. Loading efficiency was determined by RP-HPLC-UV; characterization of RIT:SM systems was carried out by apparent average hydrodynamic diameter and rheological measurements as well as different analytical techniques including Trp fluorescence, UV spectroscopy, DSC, FTIR and SEM; and delivery capacity of casein micelles was determined by in vitro experiments promoting ritonavir release. Ritonavir interacted efficiently with milk proteins, especially, casein micelles, regardless of the processing pressure; however, results suggest that, at 0.1 MPa, ritonavir interacts with caseins at the micellar surface, whilst, at 300 and 500 MPa, ritonavir is integrated to the protein matrix during UHPH treatment. Likewise, in vitro experiments showed that ritonavir release from micellar casein systems is pH dependent; with a high retention of ritonavir during simulated gastric digestion and a rapid delivery under conditions simulating the small intestine environment. Skim milk powder, especially, casein micelles are potentially suitable and efficient carrier systems to develop novel milk-based and low-ethanol powder formulations of ritonavir appropriate for pediatric applications.

Design and preparation of open circuit potential biosensor for in vitro and in vivo glucose monitoring.

PubMed

Song, Yonggui; Su, Dan; Shen, Yuan; Liu, Hongyu; Wang, Li

2017-01-01

A novel open circuit potential biosensor (OCPS) composed of a working electrode and a Ag/AgCl reference electrode was designed for in vivo continuous glucose monitoring in this work. The macroporous carbon derived from kenaf stem (KSC) was used to construct a KSC microelectrode (denoted as KSCME) which was subsequently used to load glucose oxidase (GOD) as the working electrode. The resulting GOD/KSCMEs could catalyze the oxidation of glucose directly to result in changes of the open circuit potential (V oc ) of the OCPS. The V oc of OCPS was dependent on the glucose concentration, showing a linear range of 0.03-10.0 mM (R = 0.999) with a detection limit of 10 μM. In addition, the OCPS exhibited good selectivity for glucose over other common endogenous interferences. The feasibility of the proposed OCPS for glucose detection in mice skin tumors and normal tissue homogenate samples (in vitro experiment) and rat subcutaneous glucose monitoring (in vivo experiment) was also demonstrated with satisfactory results. The biosensor represents a novel example of a superficial cancer diagnostic device, and the proposed OCPS also provides new ideas for the development of a simple and highly selective device for continuous glucose sensing.

Progesterone down-regulates SLIT/ROBO expression in mouse corpus luteum.

PubMed

Zhang, Xuejing; Mi, Meiyan; Hao, Weili; Fan, Qiongying; Gao, Bulang

2017-09-01

Progesterone produced by the corpus luteum (CL) is essential for preparation, implantation and maintenance of gestation. Furthermore, progesterone plays a protective role against luteolysis in rodents. It has been reported that Slit/Robo family members expressed in the CL and involved in prostaglandin F 2α (PGF 2α ) induced luteolysis. However, the interactions between progesterone and Slits/Robos in CL are not clear. This study was designed to examine whether or not luteolysis is regulated by the interaction of progesterone and Slits/Robos in mouse CL. In the current study, we used Real-time PCR to identify the effect of progesterone on Slit2/Robo1 expression in cultured luteal cells in vitro, and the exogenous progesterone injection on mouse luteolysis and Slit/Robo expression in vivo was studied via Real-time PCR and Western bolt. Our in vitro experiment revealed that 1μM progesterone significantly decreased Slit2/Robo1 mRNA levels at 6h, 12h and 24h. Our in vivo experiment showed that the mRNA and protein levels of Slit2 and Robo1 decreased significantly 7days after progesterone supplement. These findings indicate that progesterone maintains CL function and resists luteolysis possibly through down-regulating Slit/Robo signaling pathway in the CL. Copyright © 2017 Elsevier GmbH. All rights reserved.

Meta-analysis of the relationship between dietary tannin level and methane formation in ruminants from in vivo and in vitro experiments.

PubMed

Jayanegara, A; Leiber, F; Kreuzer, M

2012-06-01

A meta-analysis was conducted to evaluate the extent to which dietary tannin level is related to methane emissions from ruminants. Data from a total of 30 experiments comprising 171 treatments were entered in a database. In vitro batch culture and in vivo measurements were distinguished as experimental approaches. With any approach, methane declined when dietary tannins increased. The in vitro approach predicted the in vivo response quite accurately. However, in vitro, the response followed a quadratic response pattern (R(2) = 0.66; lower response with increasing tannin level), whereas in vivo, this decline was linear (R(2) = 0.29). This indicates that the in vitro batch culture is of limited accuracy for estimating effects at levels >100 g tannin/kg dry matter. The large variation in methane/digestible organic matter (OM) found at low tannin levels may explain contrasting literature reports. Methane reduction with tannins was associated with a reduced apparent digestion of OM, and especially fibre, but methane/apparently digestible OM declined also. The present findings are helpful as they identified an underlying general antimethanogenic effect of tannins across tannin sources and experimental conditions, thus allowing concentrating the search on sources with satisfactory palatability and low adverse effects on animal performance. © 2011 Blackwell Verlag GmbH.

Paradoxical Growth of Candida dubliniensis Does Not Preclude In Vivo Response to Echinocandin Therapy ▿

PubMed Central

Mariné, Marçal; Pastor, F. Javier; Sahand, Ismail H.; Pontón, José; Quindós, Guillermo; Guarro, Josep

2009-01-01

Candida dubliniensis commonly shows paradoxical or trailing growth effects in vitro in the presence of echinocandins. We tested the in vitro activities of anidulafungin, caspofungin, and micafungin against clinical isolates of C. dubliniensis and evaluated the efficacy of these drugs in two murine models of systemic infection. The three echinocandins were similarly effective in the treatment of experimental disseminated infections with C. dubliniensis strains showing or not showing abnormal growth in vitro. PMID:19786599

Virulence characteristics of five new Campylobacter jejuni chicken isolates.

PubMed

Stef, Lavinia; Cean, Ada; Vasile, Aida; Julean, Calin; Drinceanu, Dan; Corcionivoschi, Nicolae

2013-12-13

Campylobacter enteritis has emerged as one of the most common forms of human diarrheal illness. In this study we have investigated the virulence potential of five new C. jejuni chicken isolates (RO14, RO19, RO24, RO29 and RO37) originated from private households in the rural regions of Banat and Transylvania in Romania. Following isolation and in vitro virulence assay, on HCT-8 cells, our results show that all the C. jejuni chicken isolates overcome the virulence abilities of the highly virulent strain C. jejuni 81-176. Motility, an important virulence factor was significantly improved in all the new chicken isolates. The ability to survive to the antimicrobial activity of the human serum, to resist to the violent attack of bile acids and to survive in the presence of synthetic antibiotics was increased in all the chicken isolates. However, these were statistically significant only for isolates RO29 and RO37. In conclusion our study shows, based on invasiveness and motility, and also on the data provided by the serum and bile resistance experiments that all the new chicken isolates are able to infect human cells, in vitro, and could potentially represent a health hazard for humans.

The protective effect of Mucuna pruriens seeds against snake venom poisoning.

PubMed

Tan, Nget Hong; Fung, Shin Yee; Sim, Si Mui; Marinello, Enrico; Guerranti, Roberto; Aguiyi, John C

2009-06-22

The seed, leaf and root of Mucuna pruriens have been used in traditional medicine for treatments of various diseases. In Nigeria, the seed is used as oral prophylactics for snakebite. To study the protective effects of Mucuna pruriens seed extract against the lethalities of various snake venoms. Rats were pre-treated with Mucuna pruriens seed extract and challenged with various snake venoms. The effectiveness of anti-Mucuna pruriens (anti-MPE) antibody to neutralize the lethalities of snake venoms was investigated by in vitro neutralization. In rats, MPE pre-treatment conferred effective protection against lethality of Naja sputatrix venom and moderate protection against Calloselasma rhodostoma venom. Indirect ELISA and immunoblotting studies showed that there were extensive cross-reactions between anti-MPE IgG and venoms from many different genera of poisonous snakes, suggesting the involvement of immunological neutralization in the protective effect of MPE pre-treatment against snake venom poisoning. In vitro neutralization experiments showed that the anti-MPE antibodies effectively neutralized the lethalities of Asiatic cobra (Naja) venoms, but were not very effective against other venoms tested. The anti-MPE antibodies could be used in the antiserum therapy of Asiatic cobra (Naja) bites.

An in vitro atomic force microscopic study of commercially available dental luting materials.

PubMed

Djordje, Antonijevic; Denis, Brajkovic; Nenadovic, Milos; Petar, Milovanovic; Marija, Djuric; Zlatko, Rakocevic

2013-09-01

The aim of this in vitro study was to compare the surface roughness parameters of four different types of dental luting agents used for cementation of implant restorations. Five specimens (8 mm high and 1 mm thick) of each cement were made using metal ring steelless molds. Atomic Force Microscope was employed to analyze different surface texture parameters of the materials. Bearing ratio analysis was used to calculate the potential microgap size between the cement and implant material and to calculate the depth of the valleys on the cement surface, while power spectral density (PSD) measurements were performed to measure the percentage of the surface prone to bacterial adhesion. Glass ionomer cement showed significantly lower value of average surface roughness then the other groups of the materials (P < 0.05) which was in line with the results of Bearing ratio analysis. On the other side, PSD analysis showed that zinc phosphate cement experience the lowest percentage of the surface which promote bacterial colonization. Glas ionomer cements present the surface roughness parameters that are less favorable for bacterial adhesion than that of zinc phosphate, resin-modified glass ionomer and resin cements. Copyright © 2013 Wiley Periodicals, Inc.

High molecular weight chitosan derivative polymeric micelles encapsulating superparamagnetic iron oxide for tumor-targeted magnetic resonance imaging

PubMed Central

Xiao, Yunbin; Lin, Zuan Tao; Chen, Yanmei; Wang, He; Deng, Ya Li; Le, D Elizabeth; Bin, Jianguo; Li, Meiyu; Liao, Yulin; Liu, Yili; Jiang, Gangbiao; Bin, Jianping

2015-01-01

Magnetic resonance imaging (MRI) contrast agents based on chitosan derivatives have great potential for diagnosing diseases. However, stable tumor-targeted MRI contrast agents using micelles prepared from high molecular weight chitosan derivatives are seldom reported. In this study, we developed a novel tumor-targeted MRI vehicle via superparamagnetic iron oxide nanoparticles (SPIONs) encapsulated in self-aggregating polymeric folate-conjugated N-palmitoyl chitosan (FAPLCS) micelles. The tumor-targeting ability of FAPLCS/SPIONs was demonstrated in vitro and in vivo. The results of dynamic light scattering experiments showed that the micelles had a relatively narrow size distribution (136.60±3.90 nm) and excellent stability. FAPLCS/SPIONs showed low cytotoxicity and excellent biocompatibility in cellular toxicity tests. Both in vitro and in vivo studies demonstrated that FAPLCS/SPIONs bound specifically to folate receptor-positive HeLa cells, and that FAPLCS/SPIONs accumulated predominantly in established HeLa-derived tumors in mice. The signal intensities of T2-weighted images in established HeLa-derived tumors were reduced dramatically after intravenous micelle administration. Our study indicates that FAPLCS/SPION micelles can potentially serve as safe and effective MRI contrast agents for detecting tumors that overexpress folate receptors. PMID:25709439

In Vitro Comparison of Cytotoxicity of Four Root Canal Sealers on Human Gingival Fibroblasts

PubMed Central

Konjhodzic-Prcic, Alma; Gorduysus, Omer; Kucukkaya, Selen; Atila, Burcu; Muftuoglu, Sevda; Zeybek, Dilara

2015-01-01

The goal of this in vitro study was to evaluate the relative biocompatibility of four endodontic sealers on the cell culture of the human fibroblast through cytotoxicity. Materials and Methods: In this study four endodontics sealers was used GuttaFlow (Roeko)silicone based sealer, AH plus (De Tray-DENTSPLY) epoxy resin based, Apexit (Vivadent) calcium hydroxide based and Endorez (Ultradent) methacrylate based sealer. Sealers were tested on primary cell lines of human gingival fibroblasts. Experiments were preformed in laboratories of Hacettepe University of Ankara, Turkey and Faculty of Dentistry, University of Sarajevo, Bosnia and Herzegovina Cytotoxicity was determinate using WST-1 assay. Results: Results were analyzed by SPSS 19 program. Kolgomorov-Smirnov test, Shapiro-Wilk and descriptive statistics also were used, as well as Kriskall-Wallis, ANOVA test and T- test. According to our results all four sealers showed different cytotoxicity effects on human gingival fibroblast cell culture, but all of them are slightly cytotoxic. Conclusions: According to results of this study it can be concluded: all four sealers showed different cytotoxicity effects on primary cell lines of human gingival fibroblasts, but all of them are slightly cytotoxicity. PMID:25870472

High molecular weight chitosan derivative polymeric micelles encapsulating superparamagnetic iron oxide for tumor-targeted magnetic resonance imaging.

PubMed

Xiao, Yunbin; Lin, Zuan Tao; Chen, Yanmei; Wang, He; Deng, Ya Li; Le, D Elizabeth; Bin, Jianguo; Li, Meiyu; Liao, Yulin; Liu, Yili; Jiang, Gangbiao; Bin, Jianping

2015-01-01

Magnetic resonance imaging (MRI) contrast agents based on chitosan derivatives have great potential for diagnosing diseases. However, stable tumor-targeted MRI contrast agents using micelles prepared from high molecular weight chitosan derivatives are seldom reported. In this study, we developed a novel tumor-targeted MRI vehicle via superparamagnetic iron oxide nanoparticles (SPIONs) encapsulated in self-aggregating polymeric folate-conjugated N-palmitoyl chitosan (FAPLCS) micelles. The tumor-targeting ability of FAPLCS/SPIONs was demonstrated in vitro and in vivo. The results of dynamic light scattering experiments showed that the micelles had a relatively narrow size distribution (136.60±3.90 nm) and excellent stability. FAPLCS/SPIONs showed low cytotoxicity and excellent biocompatibility in cellular toxicity tests. Both in vitro and in vivo studies demonstrated that FAPLCS/SPIONs bound specifically to folate receptor-positive HeLa cells, and that FAPLCS/SPIONs accumulated predominantly in established HeLa-derived tumors in mice. The signal intensities of T2-weighted images in established HeLa-derived tumors were reduced dramatically after intravenous micelle administration. Our study indicates that FAPLCS/SPION micelles can potentially serve as safe and effective MRI contrast agents for detecting tumors that overexpress folate receptors.

Iron regulates expression of Bacillus cereus hemolysin II via global regulator Fur.

PubMed

Sineva, Elena; Shadrin, Andrey; Rodikova, Ekaterina A; Andreeva-Kovalevskaya, Zhanna I; Protsenko, Alexey S; Mayorov, Sergey G; Galaktionova, Darya Yu; Magelky, Erica; Solonin, Alexander S

2012-07-01

The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression.

Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo

PubMed Central

Bande, Omprakash; Abu El Asrar, Rania; Braddick, Darren; Dumbre, Shrinivas; Pezo, Valérie; Schepers, Guy; Pinheiro, Vitor B; Lescrinier, Eveline; Holliger, Philipp; Marlière, Philippe; Herdewijn, Piet

2015-01-01

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoCMe) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)–DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoCMe bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoCMe misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoCMe/isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality. PMID:25684598

Preparation of ritonavir nanosuspensions by microfluidization using polymeric stabilizers: I. A Design of Experiment approach.

PubMed

Karakucuk, Alptug; Celebi, Nevin; Teksin, Zeynep Safak

2016-12-01

The objective of this study was to prepare ritonavir (RTV) nanosuspensions, an anti-HIV protease inhibitor, to solve its poor water solubility issues. The microfluidization method with a pre-treatment step was used to obtain the nanosuspensions. Design of Experiment (DoE) approach was performed in order to understand the effect of the critical formulation parameters which were selected as polymer type (HPMC or PVP), RTV to polymer ratio, and number of passes. Interactions between the formulation variables were evaluated according to Univariate ANOVA. Particle size, particle size distribution and zeta potential were selected as dependent variables. Scanning electron microscopy, X-ray powder diffraction, and differential scanning calorimetry were performed for the in vitro characterization after lyophilization of the optimum nanosuspension formulation. The saturation solubility was examined in comparison with coarse powder, physical mixture and nanosuspension. In vitro dissolution studies were conducted using polyoxyethylene 10 lauryl ether (POE10LE) and biorelevant media (FaSSIF and FeSSIF). The results showed nanosuspensions were partially amorphous and spherically shaped with particle sizes ranging from 400 to 600nm. Moreover, 0.1-0.4 particle size distribution and about -20mV zeta potential values were obtained. The nanosuspension showed a significantly increased solubility when compared to coarse powder (3.5 fold). Coarse powder, physical mixture, nanosuspension and commercial product dissolved completely in POE10LE; however, cumulative dissolved values reached ~20% in FaSSIF for the commercial product and nanosuspension. The nanosuspension showed more than 90% drug dissolved in FeSSIF compared to the commercial product which showed ~50% in the same medium. It was determined that RTV dissolution was increased by nanosuspension formulation. We concluded that DoE approach is useful to develop nanosuspension formulation to improve solubility and dissolution rate of RTV. Copyright © 2016 Elsevier B.V. All rights reserved.

Responses of antioxidant enzymes, photosynthetic pigments and carbohydrates in micropropagated Pitcairnia encholirioides L.B. Sm. (Bromeliaceae) under ex vitro water deficit and after rehydration.

PubMed

Resende, C F; Pacheco, V S; Dornellas, F F; Oliveira, A M S; Freitas, J C E; Peixoto, P H P

2018-03-22

In this study, the activities of antioxidant enzymes, photosynthetic pigments, proline and carbohydrate contents in Pitcairnia encholirioides under ex vitro conditions of water deficit were evaluated. Results show that plants under progressive water stress, previously in vitro cultured in media supplemented with 30 g L-1 sucrose and GA3, accumulated more proline and increased peroxidase (POD) activity and the contents of photosynthetic pigments and carbohydrates. For plants previously in vitro cultured with 15 g L-1 sucrose and NAA, no differences were found for proline content and there were reductions in activities of peroxidase (POD), catalase (CAT) and poliphenoloxidase (PPO), and in contents of carbohydrates, with progress of ex vitro water deficit. After rehydration, plants showed physiological recovery, with enzymatic activities and contents of metabolites similar to those found in the controls not submitted to dehydration, regardless of the previous in vitro culture conditions. These results show that micropropagated P. encholirioides has high tolerance to dehydration once in ex vitro conditions, which can ensure the survival of plants from tissue culture when transferred to its natural environment, emphasizing the importance of such biotechnology for the propagation of endangered species.

Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms.

PubMed

Dezaki, Ebrahim Saedi; Yaghoubi, Mohammad Mehdi; Spiliotis, Markus; Boubaker, Ghalia; Taheri, Elham; Almani, Pooya Ghaseminejad; Tohidi, Farideh; Harandi, Majid Fasihi; Gottstein, Bruno

2016-11-01

Parts of the natural life cycle of Echinococcus granulosus can be retraced in vitro such as the development of protoscoleces into semiadult worms with three or more proglottids, or the redifferentiation of in vitro cultured protoscoleces into metacestode-like cystic structures. Most in vitro generated samples share-at the microscopical level-high similarities with those naturally grown, but developmental differences have also been documented, such as missing egg production in in vitro grown adults or unusual bladder/vesicle formation in protoscoleces cultured into the metacestode direction. The aim of the present study was to explore how far different in vitro generated stage-specific materials/structures match the natural situation on the transcriptome level, based on testing five exemplarily chosen different genes: the frizzled receptor eg-fz4 (posterior marker), the FGF receptor-like factor eg-fgfrl (anterior association), the cell differentiation protein eg-rcd1 (part of the CCR4-NOT complex, a key regulator of eukaryotic gene expression), the rapidly accelerated fibrosarcoma serin/threonin kinase eg-braf (part of the MAPK pathway involved, e.g., in EGF signaling) and the co-smad eg-smadD (downstream factor of TGFβ/BMP2/activin signaling). These genes-tested via qPCR-were selected such as to allow a discussion on their potential role in the development of E. granulosus into the adult stage. Thus, testing took place with three ex vivo isolated samples, namely (i) egg-containing adult worms, (ii) invaginated protoscoleces, and (iii) protoscolex-free germinal layer tissue. Respective data were compared (a) with in vitro generated metacestode-like microcysts developed from protoscolices, and (b) different development stages of protoscoleces in vitro cultured toward adult maturation. As a finding, only eg-smadD and partially eg-fz4 showed high expression similarities between ex vivo harvested and in vitro cultured E. granulosus, thus suggesting a putative role in adult maturation. Conclusively, the fact of using "only" five genes did not allow answering the question if ex vivo and in vitro materials are similar on the transcriptome level. Another experimental restriction arises from different growth conditions of the in vitro cultured materials, and comparing these to the ex vivo harvested ones. Future experiments may solve the problems by using fully standardized E. granulosus sample collection and fully standardized culture conditions.

Continuous glucose monitoring microsensor with a nanoscale conducting matrix and redox mediator

NASA Astrophysics Data System (ADS)

Pesantez, Daniel

The major limiting factor in kidney clinical transplantation is the shortage of transplantable organs. The current inability to distinguish viability from non-viability on a prospective basis represents a major obstacle in any attempt to expand organ donor criteria. Consequently, a way to measure and monitor a relevant analyte to assess kidney viability is needed. For the first time, the initial development and characterization of a metabolic microsensor to assess kidney viability is presented. The rate of glucose consumption appears to serve as an indicator of kidney metabolism that may distinguish reversible from irreversible kidney damage. The proposed MetaSense (Metabolic Sensor) microdevice would replace periodic laboratory diagnosis tests with a continuous monitor that provides real-time data on organ viability. Amperometry, a technique that correlates an electrical signal with analyte concentration, is used as a method to detect glucose concentrations. A novel two-electrode electrochemical sensing cell design is presented. It uses a modified metallic working electrode (WE) and a bare metallic reference electrode (RE) that acts as a pseudo-reference/counter electrode as well. The proposed microsensor has the potential to be used as a minimally invasive sensor for its reduced number of probes and very small dimensions achieved by micromachining and lithography. In order to improve selectivity of the microdevice, two electron transfer mechanisms or generations were explored. A first generation microsensor uses molecular oxygen as the electron acceptor in the enzymatic reaction and oxidizes hydrogen peroxide (H2O2) to get the electrical signal. The microsensor's modified WE with conductive polymer polypyrrole (PPy) and corresponding enzyme glucose oxidase (GOx) immobilized into its matrix, constitutes the electrochemical detection mechanism. Photoluminescence spectroscopic analysis confirmed and quantified enzyme immobilized concentrations within the matrix. In vitro testing for glucose shows increasing current with increasing analyte concentration. Testing the glucose microsensor with known concentrations of glucose over a period of 48 hours demonstrated both the potential durability and sensitivity of the device. Unknown/blind in vitro glucose experiments showed the reproducibility and accuracy of the microsensor to detect various glucose levels. Thinner polymer matrix films lead to better sensing performance during in vitro tests (0.6nA/mM lower limit sensitivity and 0.2nA/mM upper limit sensitivity). In vitro experiments using electroactive ascorbic acid (AA) and uric acid (UA) showed the selectivity of the sensor for glucose. In an effort to reduce the sensor's oxidation potential (0.7V) and noise, a second generation electron transfer approach was developed by incorporating into a modified Platinum WE with a nanoscale PPy and GOx matrix, a redox mediator. Ferrocene (Fc) was selected as the artificial electron carrier, substituting molecular oxygen in the enzymatic reaction. The incorporation of Fc into the polymer matrix is done by a simple electrochemical synthesis. Modifications in the microsensor design, materials and fabrication process are presented. Experiments with the new sensor generation resulted in higher sensitivity values (22.8nA/mM lower limit sensitivity and 12.5nA/mM upper limit sensitivity) for glucose and noise was further eliminated by operating the sensor at a lower oxidation potential (0.3V). The final experimental work consisted of preliminary ex vivo tests with the MetaSense microdevice on bovine kidney samples, which showed a qualitatively correlation between glucose consumption trend profile during preservation and viability histology outcome.

CHARACTERISTICS OF GROWTH OF SARCOMA AND CARCINOMA CULTIVATED IN VITRO

PubMed Central

Lambert, Robert A.; Hanes, Frederic M.

1911-01-01

1. The transplantable sarcomata of rats and mice grow very readily by the method of cultivating tissues in vitro. 2. Sarcomatous tissue grows in conformity to a type which may be regarded as characteristic for tissues of mesenchymal origin. 3. The growth of sarcoma cells in vitro consists in ameboid wandering into the surrounding plasma, karyokinetic proliferation. and evidences of active metabolism on the part of the cells. 4. Mouse carcinomata can be cultivated in vitro. The outgrowth of carcinoma cells assumes a sheet-like form, only one cell in thickness. They migrate into the plasma by ameboid movement, the advancing edge showing numerous prolongations of the cytoplasm into pseudopods. 5. Karyokinetic figures are frequently seen in growing carcinoma cells. The cells show evidences of active metabolism. 6. Both sarcoma and carcinoma cells cultivated in vitro show active phagocytosis; carmin particles placed in the plasma are taken up rapidly by the growing cells. PMID:19867430

Ex vivo blood vessel bioreactor for analysis of the biodegradation of magnesium stent models with and without vessel wall integration

PubMed Central

Wang, Juan; Liu, Lumei; Wu, Yifan; Maitz, Manfred F.; Wang, Zhihong; Koo, Youngmi; Zhao, Ansha; Sankar, Jagannathan; Kong, Deling; Huang, Nan; Yun, Yeoheung

2017-01-01

Current in vitro models fail in predicting the degradation rate and mode of magnesium (Mg) stents in vivo. To overcome this, the microenvironment of the stent is simulated here in an ex vivo bioreactor with porcine aorta and circulating medium, and compared with standard static in vitro immersion and with in vivo rat aorta models. In ex vivo and in vivo conditions, pure Mg wires were exposed to the aortic lumen and inserted into the aortic wall to mimic early- and long-term implantation, respectively. Results showed that: 1) Degradation rates of Mg were similar for all the fluid diffusion conditions (in vitro static, aortic wall ex vivo and in vivo); however, Mg degradation under flow condition (i.e. in the lumen) in vivo was slower than ex vivo; 2) The corrosion mode in the samples can be mainly described as localized (in vitro), mixed localized and uniform (ex vivo), and uniform (in vivo); 3) Abundant degradation products (MgO/Mg(OH)2 and Ca/P) with gas bubbles accumulated around the localized degradation regions ex vivo, but a uniform and thin degradation product layer was found in vivo. It is concluded that the ex vivo vascular bioreactor provides an improved test setting for magnesium degradation between static immersion and animal experiments and highlights its promising role in bridging degradation behavior and biological response for vascular stent research. PMID:28013101

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

PubMed

Vickery, Christopher R; Cardenas, Javier; Bowman, Marianne E; Burkart, Michael D; Da Silva, Nancy A; Noel, Joseph P

2018-06-01

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts. © 2018 Wiley Periodicals, Inc.

In Vitro Inhibitory Effect of Berberis vulgaris (Berberidaceae) and Its Main Component, Berberine against Different Leishmania Species.

PubMed

Mahmoudvand, Hossein; Sharififar, Fariba; Sharifi, Iraj; Ezatpour, Behrouz; Fasihi Harandi, Majid; Makki, Mahsa Sadat; Zia-Ali, Naser; Jahanbakhsh, Sareh

2014-03-01

Leishmaniasis has been identified as a major public health problem in tropical and sub-tropical countries. The present study was aimed to investigate antileishmanial effects of various extracts of Berberis vulgaris also its active compoenent, berberine against Leishmania tropica and L. infantum species on in vitro experiments. In this study in vitro antileishmanial activity of various extracts of B. vulgaris also its active compoenent, berberine against promastigote and amastigote stages of L. tropica and L. infantum was evaluated, using MTT assay and in a macrophage model, respectively. Furthermore, infectivity rate and cytotoxicity effects of B. vulgaris and berberine in murine macrophage cells were investigated. The findings of optical density (OD) and IC50 indicated that B. vulgaris particulary berberine significantly (P<0.05) inhibited the growth rate of promastigote stage of L.tropica and L.infantum in comparison to meglumine antimoniate (MA). In addition, B. vulgaris and berberine significantly (P<0.05) decreased the mean number of amastigotes in each macrophage as compared with positive control. In the evaluation of cytotoxicity effects, it could be observed that berberine as compared with B. vulgaris exhibited more cytotoxicity against murine macrophages. Results also showed that when parasites were pre-incubated with B. vulgaris their ability to infect murine macrophages was significantly decreased. B.vulgaris particularly berberine exhibited potent in vitro leishmanicidal effects against L. tropica and L.infantum. Further works are required to evaluate the antileishmanial effects of B.vulgaris on Leishmania species using clinical settings.

The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds.

PubMed

Ren, Tianbin; Ren, Jie; Jia, Xiaozhen; Pan, Kefeng

2005-09-15

Highly porous scaffolds of poly(lactide-co-glycolide) (PLGA) were prepared by solution-casting/salt-leaching method. The in vitro degradation behavior of PLGA scaffold was investigated by measuring the change of normalized weight, water absorption, pH, and molecular weight during degradation period. Mesenchymal stem cells (MSCs) were seeded and cultured in three-dimensional PLGA scaffolds to fabricate in vitro tissue engineering bone, which was investigated by cell morphology, cell number and deposition of mineralized matrix. The proliferation of seeded MSCs and their differentiated function were demonstrated by experimental results. To compare the reconstructive functions of different groups, mandibular defect repair of rabbit was made with PLGA/MSCs tissue engineering bone, control PLGA scaffold, and blank group without scaffold. Histopathologic methods were used to estimate the reconstructive functions. The result suggests that it is feasible to regenerate bone tissue in vitro using PLGA foams with pore size ranging from 100-250 microm as scaffolding for the transplantation of MSCs, and the PLGA/MSCs tissue engineering bone can greatly promote cell growth and have better healing functions for mandibular defect repair. The defect can be completely recuperated after 3 months with PLGA/MSCs tissue engineering bone, and the contrastive experiments show that the defects could not be repaired with blank PLGA scaffold. PLGA/MSCs tissue engineering bone has great potential as appropriate replacement for successful repair of bone defect. (c) 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005.

Biological control of Rhizoctonia solani on potato by using indigenous Trichoderma spp.

NASA Astrophysics Data System (ADS)

Durak, Emre Demirer

2016-04-01

At this study, it was aimed to determine the effect of Trichoderma isolates that was isolated from the soil samples taken from the different regions on black scurf and stem canker disease caused by Rhizoctonia solani Kühn that has been one of the biggest problems of the potato cultivation. At the end of the soil isolations, totally 81 Trichoderma isolates were obtained and their species were identified. Of these isolates, T. harzianum (42%), T. virens (31%), T. asperellum (15%) and T. viride (12%). All of the isolates were tested in vitro for their antagonistic activity against the R. solani isolate. The isolates that show high inhibition rate was selected and tested against R. solani in vitro. Potato plants were grown in a greenhouse for about 10 weeks. Then the plants were evaluated according to the scale, plant height, shoot fresh and dry weights, root fresh and dry weights were noted. The experiment was conducted two times in three replications. At the in vitro tests, generally, it was determined that Trichoderma isolates have inhibited to R. solani and in vivo, they were reduced the effects of the disease and they were raised the development of the plant. In particular, it was determined that some isolates of the T. harzianum and T. virens have reduced the severity of the disease. It was determined that both in vitro and in vivo isolates have shown different efficiency against R. solani.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma.

PubMed

Fisher, T; Golan, H; Schiby, G; PriChen, S; Smoum, R; Moshe, I; Peshes-Yaloz, N; Castiel, A; Waldman, D; Gallily, R; Mechoulam, R; Toren, A

2016-03-01

Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma

PubMed Central

Fisher, T.; Golan, H.; Schiby, G.; PriChen, S.; Smoum, R.; Moshe, I.; Peshes-Yaloz, N.; Castiel, A.; Waldman, D.; Gallily, R.; Mechoulam, R.; Toren, A.

2016-01-01

Background Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. Methods We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ9-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Results Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Conclusions Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl. PMID:27022310

Promoting effect of borneol on the permeability of puerarin eye drops and timolol maleate eye drops through the cornea in vitro.

PubMed

Wu, Chun-jie; Huang, Qin-wan; Qi, Hong-yi; Guo, Ping; Hou, Shi-xiang

2006-09-01

Studies on the influence of borneol on the penetration of puerarin eye drops and timolol maleate eye drops through the cornea, and evaluation of the ocular irritability were conducted to provide a theoretical basis for the application of borneol in enhancing corneal permeability. The cornea penetrative experiment in vitro was conducted to observe the quantitative change of puerarin and timolol maleate penetrated through the cornea after administering different dosages of borneol. The corneal hydration level and blinking frequency were recorded as irritability indexes in vitro and in vivo. The steady-flow J of high, middle and low dosage groups of puerarin eye drops with borneol were increased by 49%, 32%, 5% respectively, and permeability parameter Kp increased by 49%, 32%, 5% respectively, as compared to that of the control group. The steady-flow J of high dosage group of timolol maleate eye drops with borneol was increased by 5%; middle and low dosage groups with borneol were decreased by 6%, 3% respectively. The permeability parameter Kp of high dosage group increased by 5%, while middle and low dosage groups with borneol were decreased by 6%, 3% respectively, as compared to that of the control group. Evaluation showed no ocular irritability caused by borneol. The results of this study suggest that the promoting effect of borneol on the permeability of drugs through the cornea in vitro is selective, which indicates that borneol has the potential to be used as an ophthalmic penetration enhancer.

Genotoxic and Antigenotoxic Assessment of Chios Mastic Oil by the In Vitro Micronucleus Test on Human Lymphocytes and the In Vivo Wing Somatic Test on Drosophila.

PubMed

Vlastos, Dimitris; Drosopoulou, Elena; Efthimiou, Ioanna; Gavriilidis, Maximos; Panagaki, Dimitra; Mpatziou, Krystalenia; Kalamara, Paraskevi; Mademtzoglou, Despoina; Mavragani-Tsipidou, Penelope

2015-01-01

Chios mastic oil (CMO), the essential oil derived from Pistacia lentiscus (L.) var. chia (Duham), has generated considerable interest because of its antimicrobial, anticancer, antioxidant and other beneficial properties. In the present study, the potential genotoxic activity of CMO as well as its antigenotoxic properties against the mutagenic agent mitomycin-C (MMC) were evaluated by employing the in vitro Cytokinesis Block MicroNucleus (CBMN) assay and the in vivo Somatic Mutation And Recombination Test (SMART). In the in vitro experiments, lymphocytes were treated with 0.01, 0.05 and 0.10% (v/v) of CMO with or without 0.05 μg/ml MMC, while in the in vivo assay Drosophila larvae were fed with 0.05, 0.10, 0.50 and 1.00% (v/v) of CMO with or without 2.50 μg/ml MMC. CMO did not significantly increase the frequency of micronuclei (MN) or total wing spots, indicating lack of mutagenic or recombinogenic activity. However, the in vitro analysis suggested cytotoxic activity of CMO. The simultaneous administration of MMC with CMO did not alter considerably the frequencies of MMC-induced MN and wing spots showing that CMO doesn't exert antigenotoxic or antirecombinogenic action. Therefore, CMO could be considered as a safe product in terms of genotoxic potential. Even though it could not afford any protection against DNA damage, at least under our experimental conditions, its cytotoxic potential could be of interest.

Preparation and characterization of poly(ε-caprolactone) nanospheres containing the local anesthetic lidocaine.

PubMed

Ramos Campos, Estefânia Vangelie; Silva de Melo, Nathalie Ferreira; Guilherme, Viviane Aparecida; de Paula, Eneida; Rosa, André Henrique; de Araújo, Daniele Ribeiro; Fraceto, Leonardo Fernandes

2013-01-01

The objective of this work was to develop a modified release system for the local anesthetic lidocaine (LDC), using poly(ε-caprolactone) (PCL) nanospheres (NSs), to improve the pharmacological properties of the drug when administered by the infiltration route. In vitro experiments were used to characterize the system and investigate the release mechanism. The NSs presented a polydispersion index of 0.072, an average diameter of 449.6 nm, a zeta potential of -20.1 mV, and an association efficiency of 93.3%. The release profiles showed that the release of associated LDC was slower than that of the free drug. Atomic force microscopy analyses showed that the spherical structure of the particles was preserved as a function of time, as well as after the release experiments. Cytotoxicity and pharmacological tests confirmed that association with the NSs reduced the toxicity of LDC, and prolonged its anesthetic action. This new formulation could potentially be used in applications requiring gradual anesthetic release, especially dental procedures. Copyright © 2012 Wiley Periodicals, Inc.

Numerical Study on Focusing of Ultrasounds in Microbubble-enhanced HIFU

NASA Astrophysics Data System (ADS)

Matsumoto, Yoichiro; Okita, Kohei; Takagi, Shu

2011-11-01

The injection of microbubbles into the target tissue enhances tissue heating in High-Intensity Focused Ultrasound therapy, via inertial cavitation. The control of the inertial cavitation is required to achieve the efficient tissue ablation. Microbubbles between a transducer and a target disturb the ultrasound propagation depending on the conditions. A method to clear such microbubbles has been proposed by Kajiyama et al. [Physics Procedia 3 (2010) 305-314]. In the method, the irradiation of intense ultrasounds with a burst waveform fragmentize microbubbles in the pathways before the irradiation of ultrasounds for tissue heating. The vitro experiment using a gel containing microbubbles has showed that the method enables to heat the target correctly by controlling the microbubble distribution. Following the experiment, we simulate the focusing of ultrasounds through a mixture containing microbubbles with considering the size and number density distributions in space. The numerical simulation shows that the movement of the heating region from the transducer side to the target by controlling the microbubble distributions. The numerical results elucidate well the experimental ones.

Doxorubicin Loaded Chitosan-W18 O49 Hybrid Nanoparticles for Combined Photothermal-Chemotherapy.

PubMed

Yuan, Shanmei; Hua, Jisong; Zhou, Yinyin; Ding, Yin; Hu, Yong

2017-08-01

Combined treatment is more effective than single treatment against most forms of cancer. In this work, doxorubicin loaded chitosan-W 18 O 49 nanoparticles combined with the photothermal therapy and chemotherapy are fabricated through the electrostatic interaction between positively charged chitosan and negatively charged W 18 O 49 nanoparticles. The in vitro and in vivo behaviors of these nanoparticles are examined by dynamic light scattering, transmission electron microscopy, cytotoxicity, near-infrared fluorescence imaging, and tumor growth inhibition experiment. These nanoparticles have a mean size around 110 nm and show a pH sensitive drug release behavior. After irradiation by the 980 nm laser, these nanoparticles show more pronounced cytotoxicity against HeLa cells than that of free doxorubicin or photothermal therapy alone. The in vivo experiments confirm that their antitumor ability is significantly improved, resulting in superior efficiency in impeding tumor growth and extension of the lifetime of mice. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis, bioactivity and preliminary biocompatibility studies of glasses in the system CaO-MgO-SiO2-Na2O-P2O5-CaF2.

PubMed

Tulyaganov, D U; Agathopoulos, S; Valerio, P; Balamurugan, A; Saranti, A; Karakassides, M A; Ferreira, J M F

2011-02-01

New compositions of bioactive glasses are proposed in the CaO-MgO-SiO(2)-Na(2)O-P(2)O(5)-CaF(2) system. Mineralization tests with immersion of the investigated glasses in simulated body fluid (SBF) at 37°C showed that the glasses favour the surface formation of hydroxyapatite (HA) from the early stages of the experiments. In the case of daily renewable SBF, monetite (CaHPO(4)) formation competed with the formation of HA. The influence of structural features of the glasses on their mineralization (bioactivity) performance is discussed. Preliminary in vitro experiments with osteoblasts' cell-cultures showed that the glasses are biocompatible and there is no evidence of toxicity. Sintering and devitrification studies of glass powder compacts were also performed. Glass-ceramics with attractive properties were obtained after heat treatment of the glasses at relatively low temperatures (up to 850°C).

Quercetin attenuates high fructose feeding-induced atherosclerosis by suppressing inflammation and apoptosis via ROS-regulated PI3K/AKT signaling pathway.

PubMed

Lu, Xue-Li; Zhao, Cui-Hua; Yao, Xin-Liang; Zhang, Han

2017-01-01

Quercetin is a dietary flavonoid compound extracted from various plants, such as apple and onions. Previous studies have revealed its anti-inflammatory, anti-cancer, antioxidant and anti-apoptotic activities. This study investigated the ability of quercetin to inhibit high fructose feeding- or LPS-induced atherosclerosis through regulating oxidative stress, apoptosis and inflammation response in vivo and in vitro experiments. 50 and 100mg/kg quercetin were used in our study, showing significant inhibitory role in high fructose-induced atherosclerosis via reducing reactive oxygen species (ROS) levels, Caspase-3 activation, inflammatory cytokines releasing, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and collagen contents as well as modulating apoptosis- and inflammation-related proteins expression. We also explored the protective effects of quercetin on atherosclerosis by phosphatidylinositide 3-kinases (PI3K)/Protein kinase B (AKT)-associated Bcl-2/Caspase-3 and nuclear factor kappa B (NF-κB) signal pathways activation, promoting AKT and Bcl-2 expression and reducing Caspase-3 and NF-κB activation. Quercetin reduced the atherosclerotic plaque size in vivo in high fructose feeding-induced mice assessed by oil red O. Also, in vitro experiments, quercetin displayed inhibitory role in LPS-induced ROS production, inflammatory response and apoptosis, which were linked with PI3K/AKT-regulated Caspase-3 and NF-κB activation. In conclusion, our results showed that quercetin inhibited atherosclerotic plaque development in high fructose feeding mice via PI3K/AKT activation regulated by ROS. Copyright © 2016. Published by Elsevier Masson SAS.

Different in vitro cellular responses to tamoxifen treatment in polydimethylsiloxane-based devices compared to normal cell culture.

PubMed

Wang, Lingyu; Yu, Linfen; Grist, Samantha; Cheung, Karen C; Chen, David D Y

2017-11-15

Cell culture systems based on polydimethylsiloxane (PDMS) microfluidic devices offer great flexibility because of their simple fabrication and adaptability. PDMS devices also make it straightforward to set up parallel experiments and can facilitate process automation, potentially speeding up the drug discovery process. However, cells grown in PDMS-based systems can develop in different ways to those grown with conventional culturing systems because of the differences in the containers' surfaces. Despite the growing number of studies on microfluidic cell culture devices, the differences in cellular behavior in PDMS-based devices and normal cell culture systems are poorly characterized. In this work, we investigated the proliferation and autophagy of MCF7 cells cultured in uncoated and Parylene-C coated PDMS wells. Using a quantitative method combining solid phase extraction and liquid chromatography mass spectrometry we developed, we showed that Tamoxifen uptake into the surfaces of uncoated PDMS wells can change the drug's effective concentration in the culture medium, affecting the results of Tamoxifen-induced autophagy and cytotoxicity assays. Such changes must be carefully analyzed before transferring in vitro experiments from a traditional culture environment to a PDMS-based microfluidic system. We also found that cells cultured in Parylene-C coated PDMS wells showed similar proliferation and drug response characteristics to cells cultured in standard polystyrene (PS) plates, indicating that Parylene-C deposition offers an easy way of limiting the uptake of small molecules into porous PDMS materials and significantly improves the performance of PDMS-based device for cell related research. Copyright © 2017 Elsevier B.V. All rights reserved.

[Optimize preparation of compound licorice microemulsion with D-optimal design].

PubMed

Ma, Shu-Wei; Wang, Yong-Jie; Chen, Cheng; Qiu, Yue; Wu, Qing

2018-03-01

In order to increase the solubility of essential oil in compound licorice microemulsion and improve the efficacy of the decoction for treating chronic eczema, this experiment intends to prepare the decoction into microemulsion. The essential oil was used as the oil phase of the microemulsion and the extract was used as the water phase. Then the microemulsion area and maximum ratio of water capacity was obtained by plotting pseudo-ternary phase diagram, to determine the appropriate types of surfactant and cosurfactant, and Km value-the mass ratio between surfactant and cosurfactant. With particle size and skin retention of active ingredients as the index, microemulsion prescription was optimized by D-optimal design method, to investigate the in vitro release behavior of the optimized prescription. The results showed that the microemulsion was optimal with tween-80 as the surfactant and anhydrous ethanol as the cosurfactant. When the Km value was 1, the area of the microemulsion region was largest while when the concentration of extract was 0.5 g·mL⁻¹, it had lowest effect on the particle size distribution of microemulsion. The final optimized formulation was as follows: 9.4% tween-80, 9.4% anhydrous ethanol, 1.0% peppermint oil and 80.2% 0.5 g·mL⁻¹ extract. The microemulsion prepared under these conditions had a small viscosity, good stability and high skin retention of drug; in vitro release experiment showed that microemulsion had a sustained-release effect on glycyrrhizic acid and liquiritin, basically achieving the expected purpose of the project. Copyright© by the Chinese Pharmaceutical Association.

Iron oxide-based conjugates for cancer theragnostics

NASA Astrophysics Data System (ADS)

Phuc Nguyen, Xuan; Tran, Dai Lam; Thu Ha, Phuong; Pham, Hong Nam; Trang Mai, Thu; Linh Pham, Hoai; Le, Van Hong; Do, Hung Manh; Bich Hoa Phan, Thi; Giang Pham, Thi Ha; Nguyen, Dac Tu; Nhung Hoang, Thi My; Lam, Khanh; Quy Nguyen, Thi

2012-09-01

In this paper we first summarize our recent research on fabrication and structure characterization of conjugates of Fe3O4 nanoparticles (MNPs) encapsulated by several organic materials such as oleic acid (OL), starch (ST), dextran (D), chitosan (CS), O-carboxymethyl chitosan (OCMCS) and the copolymer of poly(styrene-co-acrylic acid (St-co-AA)). The ferrofluids stability and toxicity were also considered. The magnetic inductive heating (MIH) curves were measured using a set up with an alternating (ac) magnetic field of strength of 40-100 Oe and frequency of 180-240 kHz. We then present new results dealing with attempting to apply the MNP/copolymer ferrofluid for treatment of Sarcoma 180 tumor. In vitro as well as ex vivo MIH experiments were carried out as preparation steps in order to estimate the proper conditions for the in vivo MIH experiment. As for the latter, we have successfully carried out the treatment of solid tumor of size around 6 × 6 mm inoculated on Swiss mice with use of a dose of 0.3-0.4 mg ml-1 ferrofluid injected subcutaneously into the tumor and field-irradiated for 30 min. Two groups of treated mice recovered in three weeks from MIH treatment three times during the first week. We finally show that curcumin loaded MNP-based conjugates showed themselves to be a potential agent for application as a bimodal contrast enhancer of magnetic resonance imaging (MRI) and fluorescence imaging. Additionally, in vitro and ex vivo studies by these two techniques evidenced that macrophage is capable of uptake and tends to carry the MNPs into a tumor.

E-cigarette vapour is not inert and exposure can lead to cell damage.

PubMed

Holliday, Richard; Kist, Ralf; Bauld, Linda

2016-03-01

In vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. The widely available cell line HaCat, a spontaneously transformed immortal keratinocyte and the HNSCC cell lines HN30 and UMSCC10B were used. Cells were exposed to nicotine-containing and nicotine-free vapour extract from two popular e-cigarette brands for periods ranging from 48 hours to eight weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapour nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing two brands of e-cigs show that at biologically relevant doses, vapourised e-cig liquids induce increased DNA strand breaks and cell death, and decreased clono- genic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects of e-cig usage, as well as whether the DNA damage shown in our study as a result of e-cig exposure will lead to mutations that ultimately result in cancer.

A novel environmental chamber for neuronal network multisite recordings.

PubMed

Biffi, E; Regalia, G; Ghezzi, D; De Ceglia, R; Menegon, A; Ferrigno, G; Fiore, G B; Pedrocchi, A

2012-10-01

Environmental stability is a critical issue for neuronal networks in vitro. Hence, the ability to control the physical and chemical environment of cell cultures during electrophysiological measurements is an important requirement in the experimental design. In this work, we describe the development and the experimental verification of a closed chamber for multisite electrophysiology and optical monitoring. The chamber provides stable temperature, pH and humidity and guarantees cell viability comparable to standard incubators. Besides, it integrates the electronics for long-term neuronal activity recording. The system is portable and adaptable for multiple network housings, which allows performing parallel experiments in the same environment. Our results show that this device can be a solution for long-term electrophysiology, for dual network experiments and for coupled optical and electrical measurements. Copyright © 2012 Wiley Periodicals, Inc.

Synthesis and anticancer activity of novel curcumin-quinolone hybrids.

PubMed

Raghavan, Saiharish; Manogaran, Prasath; Gadepalli Narasimha, Krishna Kumari; Kalpattu Kuppusami, Balasubramanian; Mariyappan, Palanivelu; Gopalakrishnan, Anjana; Venkatraman, Ganesh

2015-09-01

A number of new curcumin-quinolone hybrids were synthesised from differently substituted 3-formyl-2-quinolones and vanillin and their in vitro cytotoxicity was determined on a panel of representative cell lines (A549, MCF7, SKOV3 and H460) using MTT assay. The most potent compound 14, was analysed for its mode of action using various cell biology experiments. SKOV3 cells treated with compound 14 showed distorted cell morphology under phase contrast imaging and induction of apoptosis was confirmed by Annexin V/PE assay. Further experiments on generation of reactive oxygen species (ROS) and cell cycle analysis revealed that these hybrids induce apoptosis by ROS generation and arrest cell cycle progression in S and G2/M phase. Copyright © 2015 Elsevier Ltd. All rights reserved.

How to assess the mutagenic potential of cosmetic products without animal tests?

PubMed

Speit, Günter

2009-08-01

Animal experiments (in vivo tests) currently play a key role in genotoxicity testing. Results from in vivo tests are, in many cases, decisive for the assessment of a mutagenic potential of a test compound. The Seventh Amendment to the European Cosmetics Directive will, however, ban the European marketing of cosmetic/personal care products that contain ingredients that have been tested in animal experiments. If genotoxicity testing is solely based on the currently established in vitro tests, the attrition rate for chemicals used in cosmetic products will greatly increase due to irrelevant positive in vitro test results. There is urgent need for new and/or improved in vitro genotoxicity tests and for modified test strategies. Test strategies should consider all available information on chemistry of the test substance/the chemical class (e.g. SAR, metabolic activation and dermal adsorption). Test protocols for in vitro genotoxicity tests should be sensitive and robust enough to ensure that negative results can be accepted with confidence. It should be excluded that positive in vitro test results are due to high cytotoxicity or secondary genotoxic effects which may be thresholded and/or only occur under in vitro test conditions. Consequently, further research is needed to establish the nature of thresholds in in vitro assays and to determine the potential for incorporation of mode of action data into future risk assessments. New/improved tests have to be established and validated, considering the use of (metabolically competent) primary (skin) cells, 3D skin models and cells with defined capacity for metabolic activation (e.g. genetically engineered cell lines). The sensitivity and specificity of new and improved genotoxicity tests has to be determined by testing a battery of genotoxic and non-genotoxic chemicals. New or adapted international guidelines will be needed for these tests. The establishment of such a new genotoxicity testing strategy will take time and the new in vitro genotoxicity testing will become much more complex and will require greater mechanistic understanding to build a weight of evidence decision, which will be demanding and time-consuming. At present, no validated alternative methods for the follow-up of positive results from the standard genotoxicity battery are available and an appropriate evaluation of the mutagenic potential of cosmetic ingredients without animal experiments is therefore not possible in many cases.

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition.

PubMed

Sørli, Jorid B; Huang, Yishi; Da Silva, Emilie; Hansen, Jitka S; Zuo, Yi Y; Frederiksen, Marie; Nørgaard, Asger W; Ebbehøj, Niels E; Larsen, Søren T; Hougaard, Karin S

2018-01-01

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

PubMed

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism

PubMed Central

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-01-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. PMID:26081516

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism.

PubMed

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-09-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE-/- mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

ACE2-Independent Action Of Presumed ACE2 Activators: Studies In Vivo, Ex Vivo and In Vitro

PubMed Central

Haber, Philipp K.; Ye, Minghao; Wysocki, Jan; Maier, Christoph; Haque, Syed K.; Batlle, Daniel

2014-01-01

Angiotensin converting enzyme 2, (ACE2), is a key enzyme in the metabolism of angiotensin II. 1-[[2-(dimetilamino)ethyl]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT)and Diminazene (DIZE)have been reported to exert various organ-protective effects that have been attributed to activation of ACE2. To test the effect of these compounds we studied Ang II degradation in vivo and in vitro as well as their effect on ACE2 activity in vivo and in vitro. In a model of Ang II induced acute hypertension, blood pressure recovery was markedly enhanced by XNT (slope with XNT -3.26±0.2 vs.-1.6±0.2 mmHg/min without XNT, p<0.01). After Ang II infusion, neither plasma nor kidney ACE2 activity was affected by XNT. Plasma Ang II and Ang (1-7) levels also were not significantly affected by XNT. The blood pressure lowering effect of XNT seen in WT animals was also observed in ACE2 KO mice (slope with XNT -3.09±0.30 mmHg/min vs. -1.28±0.22 mmHg/min without XNT, p<0.001). These findings show that the blood pressure lowering effect of XNT in Ang II induced hypertension cannot be due to activation of ACE2. In vitro and ex vivo experiments in both mice and rat kidney confirmed a lack of enhancement of ACE2 enzymatic activity by XNT and DIZE. Moreover, Ang II degradation in vitro and ex vivo was unaffected by XNT and DIZE. We conclude that the biologic effects of these compounds are ACE2 independent and should not be attributed to activation of this enzyme. PMID:24446061