Nonpsychotropic plant cannabinoids, cannabidivarin (CBDV) and cannabidiol (CBD), activate and desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro: potential for the treatment of neuronal hyperexcitability

Epilepsy is the most common neurological disorder, with over 50 million people worldwide affected. Recent evidence suggests that the transient receptor potential cation channel subfamily V member 1 (TRPV1) may contribute to the onset and progression of some forms of epilepsy. Since the two nonpsychotropic cannabinoids cannabidivarin (CBDV) and cannabidiol (CBD) exert anticonvulsant activity in vivo and produce TRPV1-mediated intracellular calcium elevation in vitro, we evaluated the effects of these two compounds on TRPV1 channel activation and desensitization and in an in vitro model of epileptiform activity. Patch clamp analysis in transfected HEK293 cells demonstrated that CBD and CBDV dose-dependently activate and rapidly desensitize TRPV1, as well as TRP channels of subfamily V type 2 (TRPV2) and subfamily A type 1 (TRPA1). TRPV1 and TRPV2 transcripts were shown to be expressed in rat hippocampal tissue. When tested on epileptiform neuronal spike activity in hippocampal brain slices exposed to a Mg2+-free solution using multielectrode arrays (MEAs), CBDV reduced both epileptiform burst amplitude and duration. The prototypical TRPV1 agonist, capsaicin, produced similar, although not identical effects. Capsaicin, but not CBDV, effects on burst amplitude were reversed by IRTX, a selective TRPV1 antagonist. These data suggest that CBDV antiepileptiform effects in the Mg2+-free model are not uniquely mediated via activation of TRPV1. However, TRPV1 was strongly phosphorylated (and hence likely sensitized) in Mg2+-free solution-treated hippocampal tissue, and both capsaicin and CBDV caused TRPV1 dephosphorylation, consistent with TRPV1 desensitization. We propose that CBDV effects on TRP channels should be studied further in different in vitro and in vivo models of epilepsy.

In vitro evaluation of the fermentation properties and potential probiotic activity of Lactobacillus plantarum C4 in batch culture systems

Lactobacillus plantarum C4 has been tested in in vitro pH-controlled anaerobic faecal batch cultures as compared to Lactobacillus rhamnosus GG to determine changes caused to the composition of faecal bacteria. Effects upon major groups of the microbiota and levels of short-chain fatty acids (SCFA) were assessed over 24 h. Concomitantly, hydrophobic character and ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. Quantitative analysis of bacterial populations revealed that there was a significant increase in Lactobacillus/Enterococcus numbers in vessels with probiotic supplemented with fructooligosaccharides (FOS), compared to the negative control. L. plantarum C4 showed to have more hydrophilic behaviour and fulfilled better adhesive properties, compared to L. rhamnosus GG. Thus, L. plantarum C4 can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of SCFA.

Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro

The approach of reaggregation involves the regeneration and self-renewal of histotypical 3D spheres from isolated tissue kept in suspension culture. Reaggregated spheres can be used as tumour, genetic, biohybrid and neurosphere models. In addition the functional superiority of 3D aggregates over conventional 2D cultures developed the use of neurospheres for brain engineering of CNS diseases. Thus 3D aggregate cultures created enormous interest in mechanisms that regulate the formation of multicellular aggregates in vitro. Here we analyzed mechanisms guiding the development of 3D neurosphere cultures. Adult neural stem cells can be cultured as self-adherent clusters, called neurospheres. Neurospheres are characterised as heterogeneous clusters containing unequal stem cell sub-types. Tumour necrosis factor-alpha (TNF-alpha is one of the crucial inflammatory cytokines with multiple actions on several cell types. TNF-alpha strongly activates the canonical Nuclear Factor Kappa-B (NF- kappaB) pathway. In order to investigate further functions of TNF in neural stem cells (NSCs) we tested the hypothesis that TNF is able to modulate the motility and/or migratory behaviour of SVZ derived adult neural stem cells. We observed a significantly faster sphere formation in TNF treated cultures than in untreated controls. The very fast aggregation of isolated NSCs (<2h) is a commonly observed phenomenon, though the mechanisms of 3D neurosphere formation remain largely unclear. Here we demonstrate for the first time, increased aggregation and enhanced motility of isolated NSCs in response to the TNF-stimulus. Moreover, this phenomenon is largely dependent on activated transcription factor NF-kappaB. Both, the pharmacological blockade of NF-kappaB pathway by pyrrolidine dithiocarbamate (PDTC) or Bay11-7082 and genetic blockade by expression of a transdominant-negative super-repressor IkappaB-AA1 led to decreased aggregation.

Impact of variation in structure of condensed tannins from sainfoin (Onobrychis viciifolia) on in vitro ruminal methan production and fermentation characteristics

Our study investigated the effects of condensed tannins (CT) on rumen in vitro methane (CH4) production and fermentation characteristics by incubating lucerne in buffered rumen fluid in combination with different CT extracts at 0 (control), 40, 80 and 120 g CT/kg of substrate DM. Condensed tannins were extracted from four sainfoin accessions: Rees ‘A’, CPI63763, Cotswold Common and CPI63767. Gas production (GP) was measured using a fully automated GP apparatus with CH4 measured at distinct time points. Condensed tannins differed substantially in terms of polymer size and varied from 13 (Rees ‘A’) to 73 (CPI63767) mean degree of polymerization, but had relatively similar characteristics in terms of CT content, procyanidin: prodelphinidin (PC: PD) and cis:trans ratios. Compared to control, addition of CT from CPI63767 and CPI63763 at 80 and 120 g CT/kg of substrate DM reduced CH4 by 43% and 65%, and by 23% and 57%, respectively, after 24-h incubation. Similarly, CT from Rees ‘A’ and Cotswold Common reduced CH4 by 26% and 46%, and by 28% and 46% respectively. Addition of increasing level of CT linearly reduced the maximum rates of GP and CH4 production, and the estimated in vitro organic matter digestibility. There was a negative linear and quadratic (p < 0.01) relation between CT concentration and total volatile fatty acid (VFA) production. Inclusion of 80 and 120 g CT/kg of substrate DM reduced (p < 0.001) branched-chain VFA production and acetate: propionate ratio and was lowest for CPI63767. A decrease in proteolytic activity as indirectly shown by a change in VFA composition favouring a shift towards propionate and reduction in branched-chain VFA production varied with type of CT and was highest for CPI63767. In conclusion, these results suggest that tannin polymer size is an important factor affecting in vitro CH4 production which may be linked to the CT interaction with dietary substrate or microbial cells.

Global scale transcriptome analysis of Arabidopsis embryogenesis in vitro

Background Somatic embryogenesis (SE) in plants is a process by which embryos are generated directly from somatic cells, rather than from the fused products of male and female gametes. Despite the detailed expression analysis of several somatic-to-embryonic marker genes, a comprehensive understanding of SE at a molecular level is still lacking. The present study was designed to generate high resolution transcriptome datasets for early SE providing the way for future research to understand the underlying molecular mechanisms that regulate this process. We sequenced Arabidopsis thaliana somatic embryos collected from three distinct developmental time-points (5, 10 and 15 d after in vitro culture) using the Illumina HiSeq 2000 platform. Results This study yielded a total of 426,001,826 sequence reads mapped to 26,520 genes in the A. thaliana reference genome. Analysis of embryonic cultures after 5 and 10 d showed differential expression of 1,195 genes; these included 778 genes that were more highly expressed after 5 d as compared to 10 d. Moreover, 1,718 genes were differentially expressed in embryonic cultures between 10 and 15 d. Our data also showed at least eight different expression patterns during early SE; the majority of genes are transcriptionally more active in embryos after 5 d. Comparison of transcriptomes derived from somatic embryos and leaf tissues revealed that at least 4,951 genes are transcriptionally more active in embryos than in the leaf; increased expression of genes involved in DNA cytosine methylation and histone deacetylation were noted in embryogenic tissues. In silico expression analysis based on microarray data found that approximately 5% of these genes are transcriptionally more active in somatic embryos than in actively dividing callus and non-dividing leaf tissues. Moreover, this identified 49 genes expressed at a higher level in somatic embryos than in other tissues. This included several genes with unknown function, as well as others related to oxidative and osmotic stress, and auxin signalling. Conclusions The transcriptome information provided here will form the foundation for future research on genetic and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; the genes more highly expressed in somatic embryos than in vegetative tissues can be considered as potential candidates to validate these networks.

In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports

The aim of the present study was to investigate the effect of probiotic immobilization onto wheat grains, both wet and freeze dried, on the adhesion properties of the probiotic cells and make comparisons with wet and freeze dried free cells. Lactobacillus casei ATCC 393 and Lactobacillus plantarum NCIMB 8826 were used as model probiotic strains. The results showed satisfactory adhesion ability of free cells to a monolayer of Caco-2 cells (> 1000 CFU/100 Caco-2 cells for wet cells). Cell immobilization resulted in a significant decrease in adhesion, for both wet and freeze dried formulations, most likely because immobilized cells did not have direct access to the Caco-2 cells, but it still remained in adequate levels (> 100 CFU/100 Caco-2 cells for wet cells). No clear correlation could be observed between cell adhesion and the hydrophobicity of the bacterial cells, measured by the hexadecane adhesion assay. Most notably, immobilization enhanced the monolayer integrity of Caco-2 cells, demonstrated by a more than 2-fold increase in transepithelial electrical resistance (TEER) compared to free cells. SEM micrographs ascertained the adhesion of both immobilized and free cells to the brush border microvilli. Finally, the impact of the food matrix on the adhesion properties of probiotic bacteria and on the design of novel functional products is discussed.

The identification of genes important in pseudomonas syringae pv. phaseolicola plant colonisation using in vitro screening of transposon libraries

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction

In vitro cytotoxicity of dental alloys and cpTi obtained by casting

Background: This study aimed to compare the cytotoxicity of base-metal dental alloys and to evaluate if the casting method could influence their cytotoxicity. Methods: Disks of base-metal dental alloys were cast by two methods: plasma, under argon atmosphere, injected by vacuum-pressure; and oxygen-gas flame, injected by centrifugation, except Ti-6Al-4V and commercially pure titanium (cpTi), cast only by plasma. SCC9 cells were cultured in culture media D-MEM/Ham`s F12 supplemented, at 37 degrees C in a humidified atmosphere of 5% carbon dioxide and 95% air, on the previously prepared disks. At subconfluence in wells without disks (control), cell number and viability were evaluated. Results: In plasma method, cpTi and Ti-6Al-4V were similar to control and presented higher number of cells than all other alloys, followed by Ni-Cr. In oxygen-gas name method, all alloys presented fewer cells than control. Ni-Cr presented more cells than any other alloy, followed by Co-Cr-Mo-W which presented more cells than Ni-Cr-Ti, Co-Cr-Mo, and Ni-Cr-Be. There were no significant differences between casting methods related to cell number. Cell viability was not affected by either chemical composition or casting methods. Conclusion: cpTi and Ti-6Al-4V were not cytotoxic while Ni-Cr-Be was the most cytotoxic among tested alloys. The casting method did not affect cytotoxicity of the alloys. (c) 2007 Wiley Periodicals, Inc.

In vitro osteogenesis on fluorcanasite glass-ceramic with three different chemical compositions

This study aimed at investigating in vitro osteogenesis on three fluorcanasite glass-ceramic compositions with different solubilities (K3, K5, and K8). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passage cells were cultured on K3, K5, and K8 and on Bioglass (R) 45S5 (45S5-control). Cell adhesion was evaluated at 24 h. For proliferation and viability, cells were cultured for 1, 4, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA followed by Duncan`s test. Cell adhesion, cell proliferation, viability, total protein content, and ALP activity were not affected by fluorcanasite glass-ceramic composition and solubility. Bone-like formation was similar on all fluorcanasite-glass ceramics and was reduced compared to 45S5. The changes in the chemical composition and consequently solubility of the fluorcanasite glass-ceramics tested here did not significantly alter the in vitro osteogenesis. Further modifications of the chemical composition of the fluorcanasite glass-ceramic would be required to improve bone response, making this biomaterial a good candidate to be employed as a bone substitute.

In Vitro Evaluation of Acellular Dermal Matrix as a Three-Dimensional Scaffold for Gingival Fibroblasts Seeding

Background: Tissue engineering principles could improve the incorporation of acellular dermal matrix (ADM). The aim of this study is to verify if ADM is a suitable three-dimensional matrix for gingival fibroblasts and cancerous cells ingrowth, and also if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF), and murine melanoma cell line (B16F10) were seeded on ADM for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF, and B16F10; CGF and HGF viability; and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on the ADM surface, showing no increase in cell number over the periods of study; HGF formed a monolayer on the ADM surface in a higher number at 14 days (P<0.05); B16F10 exhibited an increase in cell number within 7 days (P<0.05), and were mainly arranged in cell aggregates on the ADM, forming a continuous layer at 14 days. A higher percentage of cells on the ADM surface (P<0.05) compared to inside was observed for all cell types. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MU) values indicated higher cell viability in samples cultured with HGF compared to CGF (P=0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non-CM was observed at 48 and 72 hours (P<0.05). Conclusions: ADM is not suitable as a three-dimensional matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can only be superficially located on ADM, and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability. J Periodontol 2011;82:293-301.

In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions

P>Aim To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. Methodology Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. Results Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in < 30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. Conclusion Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro

The effect of s-nitroso-N-acetyl-1,1-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10(-3) M SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P < 0.05) while 10(-5) M SNAP showed intermediate proportions (55%). For 10(-7) M SNAP and controls (without SNAP), MII percentages were similar (72% for both, P > 0.05), but superior to the other treatment groups (P < 0.05). Blastocyst development, however, was not affected (38% for all treatments, P < 0.05). TUNEL-positive cells in hatched blastocysts (Day 9) increased when IVM included 10(-5) M SNAP (8 v. 3 to 4 cells in the other treatments, P > 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10(-7) M SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development.

Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor

Levels of ethylene and polyamines (PAs) were measured during organogenesis of hypocotyl explants of two species of passion fruit (Passiflora cincinnata Masters and Passiflora edulis Sims f. flavicarpa Degener `FB-100`) to better understand the relationships of these regulators and their influence on cell differentiation and morphogenesis. Moreover, histological investigation of shoot ontogenesis was conducted to characterize the different events involved in cell redifferentiation and regulation of PA and ethylene levels. A delay was observed in morphogenic responses of P. edulis f. flavicarpa as compared to P. cincinnata, and these changes coincided with production of elevated levels of polyamine and ethylene levels. During differentiation, cells showed high rates of expansion and elongation, and high ethylene levels were associated with high PA levels, suggesting that the two biosynthesis pathways were highly regulated. Moreover, their interaction might be an important factor for determining cell differentiation. The addition of PAs to the culture medium did not promote organogenesis; however, the incorporation of the PA inhibitor methylglyoxal bisguanylhydrazone in the culture medium reduced shoot bud differentiation, suggesting the need to maintaining a minimum level of PAs for morphogenic events to take place.

Melatonin and the time window for the expression of the alpha 8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of alpha 8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha 8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional alpha 8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha 8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha 8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha 8 nAChR subtype in full functional form. (C) 2010 ISDN. Published by Elsevier Ltd. All rights reserved.