Oxidative Stress Induced by Zearalenone in Porcine Granulosa Cells and Its Rescue by Curcumin In Vitro

Oxidative stress (OS), as a signal of aberrant intracellular mechanisms, plays key roles in maintaining homeostasis for organisms. The occurrence of OS due to the disorder of normal cellular redox balance indicates the overproduction of reactive oxygen species (ROS) and/or deficiency of antioxidants. Once the balance is broken down, repression of oxidative stress is one of the most effective ways to alleviate it. Ongoing studies provide remarkable evidence that oxidative stress is involved in reproductive toxicity induced by various stimuli, such as environmental toxicants and food toxicity. Zearalenone (ZEA), as a toxic compound existing in contaminated food products, is found to induce mycotoxicosis that has a significant impact on the reproduction of domestic animals, especially pigs. However, there is no information about how ROS and oxidative stress is involved in the influence of ZEA on porcine granulosa cells, or whether the stress can be rescued by curcumin. In this study, ZEA-induced effect on porcine granulosa cells was investigated at low concentrations (15 μM, 30 μM and 60 μM). In vitro ROS levels, the mRNA level and activity of superoxide dismutase, glutathione peroxidase and catalase were obtained. The results showed that in comparison with negative control, ZEA increased oxidative stress with higher ROS levels, reduced the expression and activity of antioxidative enzymes, increased the intensity of fluorogenic probes 2’, 7’-Dichlorodihydrofluorescin diacetate and dihydroethidium in flow cytometry assay and fluorescence microscopy. Meanwhile, the activity of glutathione (GSH) did not change obviously following 60 μM ZEA treatment. Furthermore, the underlying protective mechanisms of curcumin on the ZEA-treated porcine granulosa cells were investigated. The data revealed that curcumin pre-treatment significantly suppressed ZEA-induced oxidative stress. Collectively, porcine granulosa cells were sensitive to ZEA, which may induce oxidative stress. The findings from this study clearly demonstrate that curcumin is effective to reduce the dysregulation of cellular redox balance on porcine granulosa cells in vitro and should be further investigated for its protective role against ZEA in animals.     

Response-Predictive Gene Expression Profiling of Glioma Progenitor Cells In Vitro

Background

High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist.

Methods

In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a) to enrich specimens for brain tumor initiating cells and (b) to confront cells with a therapeutic agent before expression profiling.

Results

As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.

Conclusion

For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.     

Improving Antibiotic Activity against Wound Pathogens with Manuka Honey In Vitro

Following the discovery of synergistic action between oxacillin and manuka honey against methicillin-resistant Staphylococcus aureus, this study was undertaken to search for further synergistic combinations of antibiotics and honey that might have potential in treating wounds. Fifteen antibiotics were tested with and without sublethal concentrations of manuka honey against each of MRSA and Pseudomonas aeruginosa using disc diffusion, broth dilution, E strip, chequerboard titration and growth curves. Five novel antibiotic and manuka honey combinations were found that improved antibacterial effectiveness in vitro and these offer a new avenue of future topical treatments for wound infections caused by these two important pathogens.     

Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.     

Oral Community Interactions of Filifactor alocis In Vitro

Filifactor alocis is a gram positive anaerobe that is emerging as an important periodontal pathogen. In the oral cavity F. alocis colonizes polymicrobial biofilm communities; however, little is known regarding the nature of the interactions between F. alocis and other oral biofilm bacteria. Here we investigate the community interactions of two strains of F. alocis with Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, organisms with differing pathogenic potential in the oral cavity. In an in vitro community development model, S. gordonii was antagonistic to the accumulation of F. alocis into a dual species community. In contrast, F. nucleatum and the type strain of F. alocis formed a synergistic partnership. Accumulation of a low passage isolate of F. alocis was also enhanced by F. nucleatum. In three species communities of S. gordonii, F. nucleatum and F. alocis, the antagonistic effects of S. gordonii superseded the synergistic effects of F. nucleatum toward F. alocis. The interaction between A. actinomycetemcomitans and F. alocis was strain specific and A. actinomycetemcomitans could either stimulate F. alocis accumulation or have no effect depending on the strain. P. gingivalis and F. alocis formed heterotypic communities with the amount of P. gingivalis greater than in the absence of F. alocis. However, while P. gingivalis benefited from the relationship, levels of F. alocis in the dual species community were lower compared to F. alocis alone. The inhibitory effect of P. gingivalis toward F. alocis was dependent, at least partially, on the presence of the Mfa1 fimbrial subunit. In addition, AI-2 production by P. gingivalis helped maintain levels of F. alocis. Collectively, these results show that the pattern of F. alocis colonization will be dictated by the spatial composition of microbial microenvironments, and that the organism may preferentially accumulate at sites rich in F. nucleatum.     

Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.     

Stimulus-Specific Adaptation at the Synapse Level In Vitro

Stimulus-specific adaptation (SSA) is observed in many brain regions in humans and animals. SSA of cortical neurons has been proposed to accumulate through relays in ascending pathways. Here, we examined SSA at the synapse level using whole-cell patch-clamp recordings of primary cultured cortical neurons of the rat. First, we found that cultured neurons had high firing capability with 100-Hz current injection. However, neuron firing started to adapt to repeated electrically activated synaptic inputs at 10 Hz. Next, to activate different dendritic inputs, electrical stimulations were spatially separated. Cultured neurons showed similar SSA properties in the oddball stimulation paradigm compared to those reported in vivo. Single neurons responded preferentially to a deviant stimulus over repeated, standard stimuli considering both synapse-driven spikes and excitatory postsynaptic currents (EPSCs). Compared with two closely placed stimulating electrodes that activated highly overlapping dendritic fields, two separately placed electrodes that activated less overlapping dendritic fields elicited greater SSA. Finally, we used glutamate puffing to directly activate postsynaptic glutamate receptors. Neurons showed SSA to two separately placed puffs repeated at 10 Hz. Compared with EPSCs, GABAa receptor-mediated inhibitory postsynaptic currents showed weaker SSA. Heterogeneity of the synaptic inputs was critical for producing SSA, with glutamate receptor desensitization participating in the process. Our findings suggest that postsynaptic fatigue contributes largely to SSA at low frequencies.     

Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro

Scope

First- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.

Methods

HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.

Results

Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [³H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.

Conclusion

Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.     

A Strontium-Modified Titanium Surface Produced by a New Method and Its Biocompatibility In Vitro

Objective

To present a new and effective method of producing titanium surfaces modified with strontium and to investigate the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces modified with strontium (Sr) for bone implant applications.

Materials and Methods

Sr-modified Ti surfaces were produced by sequential treatments with NaOH, strontium acetate, heat and water. The surface characteristics and the concentration of the Sr ions released from the samples were examined. Cell adhesion, morphology and growth were investigated using osteoblasts isolated from the calvaria of neonatal Sprague-Dawley rats. Expression of osteogenesis-related genes and proteins was examined to assess the effect of the Sr-modified Ti surfaces on osteoblasts.

Results

The modified titanium surface had a mesh structure with significantly greater porosity, and approximately5.37±0.35at.% of Sr was incorporated into the surface. The hydrophilicity was enhanced by the incorporation of Sr ions and water treatment. The average amounts of Sr released from the Sr-modified plates subjected to water treatment were slight higher than the plates without water treatment. Sr promoted cellular adhesion, spreading and growth compared with untreated Ti surfaces. The Sr-modified Ti plates also promoted expression of osteogenesis-related genes,and expression of OPN and COL-І by osteoblasts. Ti plates heat treated at 700°C showed increased bioactivity in comparison with those treated at 600°C. Water treatment upregulated the expression of osteogenesis-related genes.

Conclusions

These results show that Sr-modification of Ti surfaces may improve bioactivity in vitro. Water treatment has enhanced the response of osteoblasts. The Sr-modified Ti heat-treated at 700°C exhibited better bioactivity compared with that heated at 600°C.     

A Novel Compound from the Mushroom Cryptoporus volvatus Inhibits Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) In Vitro

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a serious contagious disease in the swine industry. At present, there are no effective control strategies against PRRSV. Thus, there is an urgent need for new treatment regimens that have efficacious antiviral activity to compensate for vaccines. The anti-infective effect of Cryptoporus volvatus has previously been demonstrated in Tradational Chinese Medicine. In this report, we expected to identify a new anti-PRRSV agent in the aqueous extract of C. volvatus, by employing a combination of modern chromatographic purification techniques and indirect immunofluorescence assay (IFA). Our results showed that C. volvatus extracts from every separation step differed in their inhibitory potency on PRRSV. One anti-PRRSV component designated as C_M-H-L-5 was isolated from water-soluble fraction of C. volvatus. The inhibition induced by C_M-H-L-5 occurred in a dose-dependent manner. C_M-H-L-5 appeared to be a low-molecular-weight polyol fragment with amide groups and carboxylic acid groups. Collectively, our findings imply that C_M-H-L-5 from the aqueous extract of C. volvatus has the potential to be used for anti-PRRSV therapy.     

Nephrotoxicity of Dietary Ochratoxin A in Broiler Chikens

Graded doses of pure ochratoxin A (0,0.5,1.0,2.0,4.0, and 8.0 mug of toxin per g of feed) were incorporated into a commercial diet which was fed to chicks from 1 day to 3 weeks of age, at which time the experiments were terminated. Growth was inhibited at 2.0 4,0, and 8.0 mug/g, whereas the kidneys were enlarged at doses of 1.0 mug/g and above. Renal function as measured by clearance of phenol red was decreased 15 and 31% by doses of 4.0 and 8.0 mug/g, respectively. Uric acid was increased 38 and 48% over the control values by doses of 4.0 and 8.0 mug/g, respectively. The plasma electrolytes Na, Cl,Ca, and K were measured; however, only K was significantly ( P smaller than 0.05) altered, showing a decrease at doses of 4.0 and 8.0 mug/g. The percentage dry weight of the kidneys decreased significantly at dose levels of 4.0 and 8.0 mug/g, indicative of edema. Histological examination of kidney sections gave the impression of edema and some tubular necrosis. Pathological changes were observed at all dose levels. These data demonstrate that ochratoxin A is a severe nephrotoxin in young broiler chickens.     

Loss of Gadkin Affects Dendritic Cell Migration In Vitro

Migration is crucial for the function of dendritic cells (DCs), which act as outposts of the immune system. Upon detection of pathogens, skin- and mucosa-resident DCs migrate to secondary lymphoid organs where they activate T cells. DC motility relies critically on the actin cytoskeleton, which is regulated by the actin-related protein 2/3 (ARP2/3) complex, a nucleator of branched actin networks. Consequently, loss of ARP2/3 stimulators and upstream Rho family GTPases dramatically impairs DC migration. However, nothing is known yet about the relevance of ARP2/3 inhibitors for DC migration. We previously demonstrated that the AP-1-associated adaptor protein Gadkin inhibits ARP2/3 by sequestering it on intracellular vesicles. Consistent with a role of Gadkin in DC physiology, we here report Gadkin expression in bone marrow-derived DCs and show that its protein level and posttranslational modification are regulated upon LPS-induced DC maturation. DCs derived from Gadkin-deficient mice were normal with regards to differentiation and maturation, but displayed increased actin polymerization. While the actin-dependent processes of macropinocytosis and cell spreading were not affected, loss of Gadkin significantly impaired DC migration in vitro, however, in vivo DC migration was unperturbed suggesting the presence of compensatory mechanisms.     

Substrate Topography Determines Neuronal Polarization and Growth In Vitro

The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.     

Effects of Prestretch on Neonatal Peripheral Nerve: An In Vitro Study

Background  Characterizing the biomechanical failure responses of neonatal peripheral nerves is critical in understanding stretch-related peripheral nerve injury mechanisms in neonates. Objective  This in vitro study investigated the effects of prestretch magnitude and duration on the biomechanical failure behavior of neonatal piglet brachial plexus (BP) and tibial nerves. Methods  BP and tibial nerves from 32 neonatal piglets were harvested and prestretched to 0, 10, or 20% strain for 90 or 300 seconds. These prestretched samples were then subjected to tensile loading until failure. Failure stress and strain were calculated from the obtained load-displacement data. Results  Prestretch magnitude significantly affected failure stress but not the failure strain. BP nerves prestretched to 10 or 20% strain, exhibiting significantly lower failure stress than those prestretched to 0% strain for both prestretch durations (90 and 300 seconds). Likewise, tibial nerves prestretched to 10 or 20% strain for 300 seconds, exhibiting significantly lower failure stress than the 0% prestretch group. An effect of prestretch duration on failure stress was also observed in the BP nerves when subjected to 20% prestretch strain such that the failure stress was significantly lower for 300 seconds group than 90 seconds group. No significant differences in the failure strains were observed. When comparing BP and tibial nerve failure responses, significantly higher failure stress was reported in tibial nerve prestretched to 20% strain for 300 seconds than BP nerve. Conclusion  These data suggest that neonatal peripheral nerves exhibit lower injury thresholds with increasing prestretch magnitude and duration while exhibiting regional differences.     

Cytotoxicity of Polyaniline Nanomaterial on Rat Celiac Macrophages In Vitro

Polyaniline nanomaterial (nPANI) is getting popular in many industrial fields due to its conductivity and stability. The fate and effect of nPANI in the environment is of paramount importance towards its technological applications. In this work, the cytotoxicity of nPANI, which was prepared by rapid surface polymerization, was studied on rat celiac macrophages. Cell viability of macrophages treated with various concentrations of nPANI and different periods ranging from 24 to 72 hours was tested by a MTT assay. Damages of nPANI to structures of macrophages were evaluated according to the exposure level of cellular reactive oxygen species (ROS) and change of mitochondrial membrane potential (MMP). We observed no significant effects of nPANI on the survival, ROS level and MMP loss of macrophages at concentrations up to 1 µg/ml. However, higher dose of nPANI (10 µg/ml or above) induced cell death, changes of ROS level and MMP. In addition, an increase in the expression level of caspase-3 protein and its activated form was detected in a Western blot assay under the high dose exposure of nPANI. All together, our experimental results suggest that the hazardous potential of nPANI on macrophages is time- and dose-dependent and high dose of nPANI can induce cell apoptosis through caspase-3 mediated pathway.