Melatonin promoted chemotaxins expression in lung epithelial cell stimulated with TNF-α

BackgroundPatients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.MethodsLung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.ResultsTNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10^-10 to 10^-6M) alone didn''t change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7,10^-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7, 10^-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.ConclusionTaken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.     

A proof of the DBRF-MEGN method, an algorithm for deducing minimum equivalent gene networks

Background

We previously developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method, which deduces the most parsimonious signed directed graphs (SDGs) consistent with expression profiles of single-gene deletion mutants. However, until the present study, we have not presented the details of the method's algorithm or a proof of the algorithm.

Results

We describe in detail the algorithm of the DBRF-MEGN method and prove that the algorithm deduces all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.

Conclusions

The DBRF-MEGN method provides all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.     

Nitric Oxide Donors Augment Interleukin-1β-Induced Vascular Endothelial Growth Factor in Airway Smooth Muscle Cells

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1β (IL-1β) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48 h) PASMC were stimulated with IL-1β alone or with NO donor, l-arginine and/or NO synthase inhibitor l-NAME for 4 and 24 h. IL-1β did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24 h. VEGF release was inhibited by l-NAME (531.8 ± 52 pg/ml), but restored and further elevated by l-arginine (1,529 ± 287 pg/ml). IL-1β up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by l-NAME (1.1-fold) and augmented by l-arginine (3.8-fold) at 4 h. Restoration of a NO pathway by l-arginine in l-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [³H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1β, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.     

High production of caffeine and related enzyme activities in callus cultures of Coffea arabica L.

Callus tissue culture of Coffea arabica L. cv Hybrido de Timor prepared from apical portions of orthotropic branches produced 49 to 92 times as much caffeine per unit weight of tissue as did the original explant. Cell-free extracts made from 42 to 54-day-old callus cultures in which active biosynthesis was occurring exhibited N-methyl-N ⁹-nucleoside hydrolase and N-methyltransferase enzyme activities. Similar cell-free extracts exhibited selective biodegradative activity in forming urea from xanthine. Biosynthetic substrate specificities are similar to those of the enzyme obtained from green coffee fruit and tea leaves, suggesting that callus cultures of C. arabica form caffeine in the same way as the coffee fruit and tea leaves.     

Somatic embryogenesis of tissue cultures of Papaver somniferum and Papaver orientale and its relationship to alkaloid and lipid metabolism

Transfer from complete to 2,4-D free Gamborg's B5-medium efficiently induced somatic embryogenesis in Papaver tissue cultures (P. somniferum and P. orientale). Embryogenesis was preceded by a strong temporary accumulation of triacylglycerols. In both tissue cultures large amounts of sanguinarine type alkaloids were present, which disappeared during regeneration in the P. orientale cultures but persisted in the P. somniferum cultures. In the P. somniferum cultures protopine and morphine type alkaloids (morphine, codeine, thebaine) appeared about 45 days after exchanging the medium. Thebaine was the main alkaloid in the P. somniferum embryoids accumulating up to 0.2 % of dry weight.     

A special issue on DNA damage response and genome stability

A novel, cancer-fighting function was recently discovered for Smad ubiquitination regulatory factor 2 (Smurf2).     

Inactivation of aphidicolin by plant cells

Plant cells are endowed with an aphidicolin inactivating activity. Data on cultured cells show that the rate of inactivation depends on the cell type, Daucus carota cells being the most effective among the other tested materials (Oryza sativa and Nicotiana plumbaginifolia). Also germinating seedling of Haplopappus gracilis and of Citrullus vulgaris inactivate aphidicolin. Inactivation, which may lead to unexpected results when a prolonged incubation with the drug is required, as in the case of the induction of synchrony of the cell cycle by aphidicolin, can be controlled by appropriately choosing the experimental conditions.     

GFT projection NMR for efficient 1H/13C sugar spin system identification in nucleic acids

A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify ¹H/¹³C sugar spin systems in ¹³C labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of ¹³C?C¹H groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1??- and 3??-CH signal dispersion for complete spin system identification including 5??-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3??-untranslated region of the pre-mRNA of human U1A protein.     

Towards a noninvasive anatomical and functional diagnostic work-up of patients with suspected coronary artery disease

Combining multidetector computed tomography and cardiovascular magnetic resonance imaging provides the clinician a strategy to comprehensively evaluate coronary morphology and function noninvasively. In the MARCC trial (Magnetic Resonance and CT in suspected CAD) a new noninvasive diagnostic work-up for patients with suspected coronary artery disease will be developed, involving the sequential use of both imaging techniques. (Neth Heart J 2010;18:270-3.)     

Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Two novel techniques improve division and colony formation from protoplasts:

1. Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.
2. Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).

The combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.     

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K⁺ or Cl^? or HCO ₃ ^? by Na⁺, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na⁺ by tetramethylammonium (TMA⁺) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10^?4 m, anthracene-9-COOH 5×10^?4 m, furosemide 5×10^?4 m or ethacrynic acid 5×10^?5 m, also abolished RVI. On the other hand, addition of either Ba²⁺ 10^?3 m, SITS 5× 10^?4 m, ouabain 10^?3 m or vanadate 10^?3 m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10^?6 m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na⁺, K⁺, Cl^?, Ca²⁺ and HCO ₃ ^? transport pathways and a ouabain-insensitive ATPase stimulated by Na⁺. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.     

Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites

Background

High throughput techniques have generated a huge set of biological data, which are deposited in various databases. Efficient exploitation of these databases is often hampered by a lack of appropriate tools, which allow easy and reliable identification of genes that miss functional characterization but are correlated with specific biological conditions (e.g. organotypic expression).

Results

We have developed a simple algorithm (DGSA = Database-dependent Gene Selection and Analysis) to identify genes with unknown functions involved in organ development concentrating on the heart. Using our approach, we identified a large number of yet uncharacterized genes, which are expressed during heart development. An initial functional characterization of genes by loss-of-function analysis employing morpholino injections into zebrafish embryos disclosed severe developmental defects indicating a decisive function of selected genes for developmental processes.

Conclusion

We conclude that DGSA is a versatile tool for database mining allowing efficient selection of uncharacterized genes for functional analysis.     

Absence of carious lesions at margins of glass-ionomer cement and amalgam restorations: An update of systematic review evidence

Background

A recent analysis of protein sequences deposited in the NCBI RefSeq database indicates that ~8.5 million protein sequences are encoded in prokaryotic and eukaryotic genomes, where ~30% are explicitly annotated as "hypothetical" or "uncharacterized" protein. Our Comparison of Protein Active-Site Structures (CPASS v.2) database and software compares the sequence and structural characteristics of experimentally determined ligand binding sites to infer a functional relationship in the absence of global sequence or structure similarity. CPASS is an important component of our Functional Annotation Screening Technology by NMR (FAST-NMR) protocol and has been successfully applied to aid the annotation of a number of proteins of unknown function.

Findings

We report a major upgrade to our CPASS software and database that significantly improves its broad utility. CPASS v.2 is designed with a layered architecture to increase flexibility and portability that also enables job distribution over the Open Science Grid (OSG) to increase speed. Similarly, the CPASS interface was enhanced to provide more user flexibility in submitting a CPASS query. CPASS v.2 now allows for both automatic and manual definition of ligand-binding sites and permits pair-wise, one versus all, one versus list, or list versus list comparisons. Solvent accessible surface area, ligand root-mean square difference, and Cβ distances have been incorporated into the CPASS similarity function to improve the quality of the results. The CPASS database has also been updated.

Conclusions

CPASS v.2 is more than an order of magnitude faster than the original implementation, and allows for multiple simultaneous job submissions. Similarly, the CPASS database of ligand-defined binding sites has increased in size by ~ 38%, dramatically increasing the likelihood of a positive search result. The modification to the CPASS similarity function is effective in reducing CPASS similarity scores for false positives by ~30%, while leaving true positives unaffected. Importantly, receiver operating characteristics (ROC) curves demonstrate the high correlation between CPASS similarity scores and an accurate functional assignment. As indicated by distribution curves, scores ≥ 30% infer a functional similarity. Software URL: http://cpass.unl.edu.     

Osteopathic manipulative treatment and its relationship to autonomic nervous system activity as demonstrated by heart rate variability: a repeated measures study

Background

Osteopathic manipulative treatment (OMT) and ultrasound physical therapy (UPT) are commonly used for chronic low back pain. Although there is evidence from a systematic review and meta-analysis that OMT generally reduces low back pain, there are no large clinical trials that specifically assess OMT efficacy in chronic low back pain. Similarly, there is a lack of evidence involving UPT for chronic low back pain.

Methods

The OSTEOPAThic Health outcomes In Chronic low back pain (OSTEOPATHIC) Trial is a Phase III randomized controlled trial that seeks to study 488 subjects between August 2006 and June 2010. It uses a 2 × 2 factorial design to independently assess the efficacy of OMT and UPT for chronic low back pain. The primary outcome is a visual analogue scale score for pain. Secondary outcomes include back-specific functioning, generic health, work disability, and satisfaction with back care.

Conclusion

This randomized controlled trial will potentially be the largest involving OMT. It will provide long awaited data on the efficacy of OMT and UPT for chronic low back pain.

Trial registration

http://www.clinicaltrials.gov, NCT00315120     

Multiscale Hy3S: Hybrid stochastic simulation for supercomputers

Background

DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell.

Results

We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100, 000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods.

Conclusion

Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

Indole alkaloids from cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga

From cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga grown under standard conditions, six monoterpenoid indole alkaloids have been isolated and identified. T. divaricata synthesized apparicine, catharanthine, coronaridine, conoflorine, tubotaiwine and vinervine, whereas T. iboga produced tubotaiwine and conoflorine. Both cultures are a reasonable source for conoflorine, which is expected to be a good candidate for studying the mechanism of Aspidosperma type alkaloid formation at the cell-free level.     

Genome skimming identifies polymorphism in tern populations and species

Background

Large-scale protein structure alignment, an indispensable tool to structural bioinformatics, poses a tremendous challenge on computational resources. To ensure structure alignment accuracy and efficiency, efforts have been made to parallelize traditional alignment algorithms in grid environments. However, these solutions are costly and of limited accessibility. Others trade alignment quality for speedup by using high-level characteristics of structure fragments for structure comparisons.

Findings

We present ppsAlign, a parallel protein structure Alignment framework designed and optimized to exploit the parallelism of Graphics Processing Units (GPUs). As a general-purpose GPU platform, ppsAlign could take many concurrent methods, such as TM-align and Fr-TM-align, into the parallelized algorithm design. We evaluated ppsAlign on an NVIDIA Tesla C2050 GPU card, and compared it with existing software solutions running on an AMD dual-core CPU. We observed a 36-fold speedup over TM-align, a 65-fold speedup over Fr-TM-align, and a 40-fold speedup over MAMMOTH.

Conclusions

ppsAlign is a high-performance protein structure alignment tool designed to tackle the computational complexity issues from protein structural data. The solution presented in this paper allows large-scale structure comparisons to be performed using massive parallel computing power of GPU.