Tumor delivery of ultrasound contrast agents using Shiga toxin B subunit

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound.     

Spectroscopic characterization of furosemide binding to human carbonic anhydrase II

This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of F?rster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding.     

Synthesis of rhamnogalacturonan I fragments by a modular design principle

The improved syntheses of methyl 2-O-acetyl-3-O-benzyl-alpha-L-rhamnopyranoside (12) and 1,2-di-O-acetyl-3-O-benzyl-alpha-L-rhamnopyranose (15), which were used as glycosyl acceptor and donor, respectively, are described. Glycosylation of the O-4 position of both rhamnose derivatives with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide (26) provided disaccharides 27 and 29. After partial deprotection of 27 and coupling of the resulting 28 with disaccharide 19, tetrasaccharide 31 was obtained. Furthermore, transforming of 29 into the corresponding bromide 30 and coupling with galacturonates 16 and 32 provided trisaccharides 33 and 34, respectively, which could be regarded as building blocks of ramified rhamnogalacturonan fragments. The preparation of tetra- (21) and hexasaccharide (25) of rhamnogalacturonan I is reported to demonstrate the feasibility of the synthesis of larger pectin fragments using the modular design principle with this type of building blocks.     

Association of Genetic Structure and Diversity in Iranian Wild Germplasms of Mentha longifolia L. Based on Phenotypical,Biochemical, and Molecular Markers

Mentha longifolia L. is well-known to be one of the most pervasive wild-growing species of the Lamiaceae family, which has extensive beneficial properties in the fields of pharmacology and biological products. In the present study, the correlation between Inter-simple sequence repeat (ISSR) markers and morpho-chemical parameters of twenty different M. longifolia accessions (MLACs) were assessed. The geographic information system (GIS) has been employed to interpret the original habitat of the accessions in Iran. ISSR analysis indicated a remarkable difference in the studied accessions, segregated them into three main groups, constructed by an unweighted pair-group method with arithmetic (UPGMA) and principal coordinate analysis (PCoA). A total of 89 bands were generated by 12 ISSR primers, among which 82 (91.97 %) of them were polymorphic. The cluster analysis based on agro-morphological data scattered MLACs into two main groups. The essential oils (EOs) were analyzed through GC/FID/MS, and four chemotypes were characterized according to the major constituents. Pulegone ranged from 0.17 to 69.50 % was the main oil constituent with the highest content. Also, HPLC-PDA was employed to identify and to quantify the phenolic compounds in the MeOH extracts of MLACs. Heatmap cluster based on phenolic compounds produced three main categories of accessions. The components identified in the extracts were rosmarinic acid, rutin, vanillic acid, ferulic acid, chlorogenic acid, caffeic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and p-coumaric acid, which among them rosmarinic acid (RA) varied from 39.16 to 261.55 mg/100 g (DW) as a predominant constituent. Subsequently, multiple regression analyses between ISSR fragments and morpho-chemical data illustrated considerable relationships in the plant materials. The high variation and correlation observed in metabolic and phenotypic traits of MLACs establish an adequate source to conduct reserves conservation programs.     

Does immobilization of Thiobacillus ferrooxidans really decrease the effect of temperature on its activity?

It has been suggested that the influence of temperature on the activity of Thiobacillus ferrooxidans is decreased when the cells are immobilized. This is contrary to normal expectations and the work presented here indicates that it is not, in fact, the case. Experimental results are presented which show that the kinetics of Fe(II) oxidation by biofilms at 30°C are significantly faster than those at 20°C. Temperature, therefore, plays an important role in the activity of T. ferrooxidans, even in the immobilized form, and results indicate that it can also be used to control the level of biomass within the immobilized cell bioreactor.     

Redescription of <Emphasis Type="Italic">Gaeolaelaps deinos</Emphasis> (Acari: Mesostigmata): an ant-associated laelapid mite

In this paper, we redescribe Gaeolaelaps deinos (Zeman 1982) based on morphological characters of female and male specimens collected from nests of Lasius sp. (Hymenoptera: Formicidae) Khuzestan and Chaharmahal va Bakhtiari Provinces, Iran, and based on its holotype photos. We also present an identification key for Gaeolaelaps aculeifer-like species group from Iran.     

Physical and Biological Treatment of Oil-Contaminated Soil in a Baffled Roller Bioreactor

Physical and biological removal of diesel oil from contaminated soil was studied in a baffled roller bioreactor. Initially, the effects of four factors (soil loading, temperature, pH, and surfactant) on physical removal of diesel oil were investigated. Only the presence of a surfactant (sodium dodecyl sulfate [SDS]) demonstrated a significant effect on diesel oil removal. Diesel oil removal efficiency was increased from 32.0% to 63.9% in the presence of 100 mg/L SDS. Using a microbial culture enriched from contaminated soil, biological treatment of diesel oil polluted soil under different soil loadings (15% to 50%), different diesel oil concentrations (1 to 50 g/L), and different types of soil (sand, silt, and clay) was then investigated in the baffled roller bioreactor. Biodegradation consisted of both fast and slow stages for degradation of light and heavy compounds, respectively. All biodegradation experiments demonstrated significant decreases in diesel oil concentrations (88.3% in 14 days for initial diesel oil concentrations of 1000 mg/L and a wide range of soil loadings). The presence of silty or sandy soils enhanced the biodegradation rate compared to the control bioreactor (without soil). The sandy soil loading had no effect on the biodegradation results. Using the enriched culture, the baffled roller bioreactor was able to biodegrade high diesel concentrations (up to 50 g/L) with biodegradation rates of 112.2 and 39.3 mg/L· h during fast and slow stages, respectively.     

The role of copper transporters in the development of resistance to Pt drugs

Recent studies in yeast, mouse and human cells suggest that the conserved metal binding transporters of the Cu homeostasis pathway can mediate resistance to Pt drugs in cancer cells. This review summarizes the data available from these studies. The observation that cells selected for resistance to Cu or the Pt drugs display bidirectional cross-resistance, parallel defects in the transport of Cu and the Pt drugs and altered expression of Cu transporters is consistent with the concept that the Cu homeostasis proteins regulate sensitivity to the Pt drugs by influencing their uptake, efflux and intracellular distribution. This model is supported by the finding that when mammalian and yeast cells are genetically engineered to express altered levels of the Cu transporters they exhibit altered sensitivity to Pt drugs and are defective in intracellular Pt accumulation due to altered uptake and/or efflux rates. Negative associations between the expression of ATP7A and ATP7B and the outcome of Pt therapy further support the significance of the Cu homeostasis proteins as both markers of and contributors to Pt resistance.     

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma

Background

Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality.

Results

Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37×10⁶-86×10⁶ unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing.

Conclusions

Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-476) contains supplementary material, which is available to authorized users.     

Impact of nitrate-mediated microbial control of souring in oil reservoirs on the extent of corrosion

The effect of microbial control of souring on the extent of corrosion was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in an SRB consortium enriched from produced water from a Canadian oil reservoir. The average corrosion rate induced by the SRB consortium (1.4 g x m(-2) x day(-1)) was faster than that observed in the presence of strain Lac6 (0.2 g x m(-2) x day(-1)). Examination of the metallic coupons at the end of the tests indicated a uniform corrosion in both cases. Addition of CVO and 10 mM nitrate to a fully grown culture of Lac6 or the SRB consortium led to complete removal of sulfide from the system and a significant increase in the population of CVO, as determined by reverse sample genome probing. In the case of the SRB consortium addition of just nitrate (10 mM) had a similar effect. When grown in the absence of nitrate, the consortium was dominated by Desulfovibrio sp. strains Lac15 and Lac29, while growth in the presence of nitrate led to dominance of Desulfovibrio sp. strain Lac3. The addition of CVO and nitrate to the Lac6 culture or nitrate to the SRB consortium accelerated the average corrosion rate to 1.5 and 2.9 g x m(-2) x day(-1), respectively. Localized corrosion and the occurrence of pitting were apparent in both cases. Although the sulfide concentration (0.5-7 mM) had little effect on corrosion rates, a clear increase of the corrosion rate with increasing nitrate concentration was observed in experiments conducted with consortia enriched from produced water.