Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface.     

Fructose-2,6-bisphosphate: a traffic signal in plant metabolism

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) regulates key reactions of the primary carbohydrate metabolism in all eukaryotes. In plants, Fru-2,6-P(2) coordinates the photosynthetic carbon flux into sucrose and starch biosynthesis. The use of transgenic plants has allowed the regulatory models to be tested by modifying the Fru-2,6-P(2) levels and the enzymes regulated by Fru-2,6-P(2). Genes for the bifunctional plant enzyme that synthesizes and degrades Fru-2,6-P(2) have been isolated and molecular characterization has provided new insight into structure and molecular regulation of the enzyme. Advances in Fru-2,6-P(2) physiology and molecular biology are discussed. These advances have not only enlightened in vivo operation of Fru-2,6-P(2) but also revealed that the Fru-2,6-P(2) regulatory system is highly complex and interacts with other regulatory mechanisms.     

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia,phylum Chloroflexi

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated ‘DEH-J10'' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO₂ or oxidation of organics completely to CO₂. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.     

BNP Predicts Chemotherapy-Related Cardiotoxicity and Death: Comparison with Gated Equilibrium Radionuclide Ventriculography

Cardiotoxicity is a dose-limiting side-effect of cancer chemotherapeutics such as anthracyclines. The drug-induced cardiac toxicity is currently monitored with repeated assessments of the left ventricular ejection fraction (LVEF) using multigated equilibrium radionuclide ventriculography (MUGA) or echocardiography. However, the plasma cardiac biomarker B-type natriuretic peptide (BNP) has been suggested for early identification of cardiac dysfunction. The aim of the study was to compare LVEF obtained by MUGA and plasma BNP as predictors of developing congestive heart failure (CHF) or death in a population of anthracycline-treated cancer patients.

Methods

We prospectively followed 333 cancer patients referred to our department for routine monitoring of LVEF with MUGA and measurement of BNP, January-December 2004. Study end points were hospitalization for CHF and death during follow-up 2004-2010. Data were obtained from the Danish National Patient Registry.

Results

During follow-up (mean 1,360 days), 21 of the patients were admitted to hospital with a diagnosis of CHF and 194 of the patients died. BNP levels were significantly higher and LVEF lower in the group of patients that developed CHF. Using cut-off points of BNP>100 pg/ml (HR 5.5; CI 1.8–17.2; p = 0.003) and LVEF <50% (HR 7.9; CI 3.0–21.4; p<0.001) both significantly predicted CHF. Using the same cut-off points only BNP (HR 1.9; CI 1.3-2.9; p = 0.002) and not LVEF (HR 1.1; CI 0.7–1.8; p = 0.58) was predictive of overall death. In multivariate Cox analysis both BNP and LVEF were independent predictors of CHF while age remained the only independent predictor of overall death.

Conclusion

In cancer patients treated with cardiotoxic chemotherapy both BNP and LVEF can significantly predict subsequent hospitalization with CHF. In addition, BNP and not LVEF has a prognostic value in detecting overall death. This prospective study based on the hitherto largest study population supports BNP as a clinical relevant method for monitoring chemotherapy-related cardiac failure and death.     

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

Flow sorting from organ material by intracellular markers.

BACKGROUND: Fluorescence-activated cell sorting (FACS) is an attractive technique for gene or protein expression studies in rare cell populations. For cell types where specific surface markers are not known, intracellular markers can be used. However, this approach is currently held to be difficult, as the required fixation and permeabilization may cause protein modification and RNA degradation. METHODS AND RESULTS: Using the rat thyroid gland as model, rare (parafollicular) and frequent (follicular) endocrine cell types were sorted based on immunostaining for intracellular calcitonin peptide and thyroglobulin protein expression. The sorted cells were compatible with Western blot analysis of proteins, immunoassay detection of calcitonin peptide hormone and RT-PCR. CONCLUSION: We developed a robust FACS protocol that allows flow sorting of rare cells from dissociated organ material, based on intracellular markers. Our FACS protocol is compatible with downstream analysis of proteins, peptides, and mRNA in the sorted cells.     

Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions.     

Immune activation in advanced cancer patients treated with recombinant IL-21: multianalyte profiling of serum proteins

Purpose  Recombinant interleukin-21 (rIL-21) is an immune stimulating cytokine recently tested in two Phase 1 trials for immune responsive cancers. A secondary objective of these trials was to characterize pharmacodynamic responses to rIL-21 in patients. Here, we report the effects of systemic rIL-21 on serum markers of immune stimulation. Experimental design  Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 μg/kg using two distinct treatment regimens: thrice weekly (‘3/w’) for 6 weeks; or once daily for five consecutive days followed by nine dose-free days (‘5 + 9’). In the absence of dose limiting toxicity, additional cycles of dosing were initiated immediately following the nine dose-free days. An array of 70 different proteins was profiled in subject serum samples from several time points during the course of the study. Hierarchical clustering analysis was performed on a normalized subset of these data. Results  Systemic administration of rIL-21 affected the serum levels of several cytokines, chemokines, acute-phase proteins and cell adhesion proteins. The magnitude and duration of response were dose dependent for a subset of these biomarkers. The 5 + 9 dosing regimen generally produced cyclic changes that were of greater magnitude, as compared to a more chronic stimulation with the 3/w dosing regimen. Despite these differences, rIL-21 effects on many analytes were similar between regimens when averaged over the time of treatment. Based on similar temporal, between-subject and dose response changes, groups of analytes were identified that exhibited distinct components of the rIL-21-mediated immune activation. Biomarkers indicative of lymphocyte activation (increased IL-16, decreased RANTES), acute phase response (increased CRP, ferritin), myeloid activation (increased MDC, MIP-1 alpha), and leukocyte chemotaxis/trafficking (increased sCAMs, MCP-1) were strongly modulated in subjects treated with rIL-21. Conclusions  Administration of rIL-21 resulted in activation of multiple cell types and immune response pathways. The changes observed in serum proteins were consistent with coincident processes of lymphoid and myeloid cell activation and trafficking, and acute phase response. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Contrasting responses of bee communities to coffee flowering at different spatial scales

While investigating biodiversity patterns on different spatial scales, ecological processes determining these patterns have been rarely analysed. Flower visitation by bees is an important ecological process that is related to floral resource availability. However, little is known about whether responses of bee communities to floral resource availability change at different spatial scales. We studied density and species richness of flower-visiting bees in relation to floral resource availability, provided by coffee, in traditional agroforestry systems on a field, shrub, and branch scale. On a field scale, mean bee density per shrub increased with decreasing proportion of flowering coffee shrubs per site, showing a dilution effect. Conversely, on shrub and branch scales bee density per shrub, or shrub part, increased with increasing number of inflorescences, showing a concentration effect. Additionally, bee density per shrub was higher on those that were only partly, rather than totally surrounded by other flowering coffee shrubs. Species richness of flower-visiting bees was positively affected by high resource availability on a shrub and a branch scale, expressed by a high number of inflorescences, but at the field scale the proportion of flowering shrubs per site did not have any effect on species richness. Our results show contrasting responses of the community of flower-visiting bees to floral resource availability, depending on the spatial scale considered. We conclude that patterns of flower-visiting bee communities of only one spatial scale can not be generalized, since the number of pollinators may be limited on a field scale, but not on smaller scales.