Development of microsatellites for the peat moss Sphagnum capillifolium using ISSR cloning

Sphagnum mosses are major components of peat bogs but populations of many species are under threat due to habitat fragmentation resulting from the cutting of peat for fuel. We have used an intersimple sequence repeat (ISSR)-based cloning method to develop nine polymorphic nuclear microsatellites for the peat moss species Sphagnum capillifolium. Between three and seven alleles per locus were detected in a sample of 48 haploid gametophytes and levels of gene diversity ranged from 0.5391 to 0.7960. These represent the first microsatellite markers developed for this important genus and most also exhibited cross-species amplification across a range of common Sphagnum species.

Development of a QSPR correlation for the parachor of 1,3-dialkyl imidazolium based ionic liquids

Ionic liquids have received significant interest from both academia and industry for a wide range of applications which often requires knowledge of their thermophysical properties. Quantitative structure-property relationship correlations and group contribution methods for thermophysical properties of ionic liquids are a basic necessity for the development of computer aided molecular design approaches for these liquids and subsequently offer the potential for designing an ionic liquid having a desirable set of thermophysical properties. However, the limited availability of experimental thermophysical data and their quality have prevented the development of such tools. Based on previously reported experimental surface tension data, a correlation of the parachors with the molar volume of the ionic liquids has been developed. The predicted parachor values have been shown to be in good agreement with the experimental data. A maximum deviation of

Rare-earth quinolinates: Infrared-emitting molecular materials with a rich structural chemistry

Near-infrared-emitting rare-earth chelates based on 8-hydroxyquinoline have appeared frequently in recent literature, because they are promising candidates for active components in near-infrared-luminescent optical devices, such as optical amplifiers, organic light-emitting diodes, .... Unfortunately, the absence of a full structural investigation of these rare-earth quinolinates is hampering the further development of rare-earth quinolinate based materials, because the luminescence output cannot be related to the structural properties. After an elaborate structural elucidation of the rare-earth quinolinate chemistry we can conclude that basically three types of structures can be formed, depending on the reaction conditions: tris complexes, corresponding to a 1:3 metal-to-ligand ratio, tetrakis complexes, corresponding to a 1:4 metal-to-ligand ratio, and trimeric complexes, with a 3:8 metal-to-ligand ratio. The intensity of the emitted near-infrared luminescence of the erbium(Ill) complexes is highest for the tetrakis complexes of the dihalogenated 8-hydroxyquinolinates.

Langerhans cells are critical in the development of atopic dermatitis-like inflammation and symptoms in mice

Genetic or vitamin D3-induced overexpression of thymic stromal lymphopoietin (TSLP) by keratinocytes results in an atopic dermatitis (AD)-like inflammatory phenotype in mice echoing the discovery of high TSLP expression in epidermis from AD patients. Although skin dendritic cells (DC) are suspected to be involved in AD, direct evidence of a pathogenetic role for skin DC in TSLP-induced skin inflammation has not yet been demonstrated. In a mouse model of AD, i.e. mice treated with the low-calcemic vitamin D3 analogue, MC903, we show that epidermal Langerhans cells (LC)-depleted mice treated with MC903 do neither develop AD-like inflammation nor increased serum IgE as compared to vitamin D3 analogue-treated control mice. Accordingly, we show that, in mice treated with MC903 or in K14-TSLP transgenic mice, expression of maturation markers by LC is increased whereas maturation of dermal DC is not altered. Moreover, only LC are responsible for the polarization of naive CD4+ T cells to a Th2 phenotype, i.e. decrease in interferon-gamma and increase in interleukin (IL)-13 production by CD4+ T cells. This effect of LC on T-lymphocytes does not require OX40-L/CD134 and is mediated by a concomitant down-regulation of IL-12 and CD70. Although it was previously stated that TSLP up-regulates the production of thymus and activation-regulated chemokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) and macrophage-derived chemokine (MDC)/CCL22 by human LC in vitro, our work shows that production of these Th2- cell attracting chemokines is increased only in keratinocytes in response to TSLP overexpression. These results demonstrate that LC are required for the development of AD in mouse models of AD involving epidermal TSLP overexpression.

Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities

BACKGROUND: The term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs). METHODS: Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. RESULTS: Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. CONCLUSIONS: This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

A retrospective analysis of spring barley germplasm development from 'foundation genotypes' to currently successful cultivars

Twenty-eight mapped barley SSRs were used to examine changes in the level and pattern of variability in northern European spring barley over time. Comparing the most recently introduced cultivars with a group of 19 landraces and key progenitors termed 'foundation genotypes' we observed a reduction in the spectrum of alleles at 28 loci over time, and highlighted chromosomal regions with limited SSR allelic variation. The 19 'foundation genotypes' contained 72% of the alleles present in all the cultivars sampled. The smallest number of genotypes required to encompass all of the alleles detected in this study was 44, several of which were recently introduced cultivars. The level of diversity within modern cultivars was lower (0.484) than in the 'foundation genotypes' (0.597), although the values varied with the SSR locus. A total of 74 rare alleles (frequency

Nutritonal status of adult ewes during early and mid-pregnancy. 1. Effects of plane of nutrition on ewe reproduction and offspring performance to weaning

The objective of this study was to determine the effects of plane of nutrition during early and mid-pregnancy on the performance of mature ewes and their offspring. From day 0 to day 39 post mating (early pregnancy, EP), 82 multiparous ewes were fed to provide either 60% (low, L), 100% (medium, M) or 200% (high, H) of predicted metabolisable energy (ME) requirements for maintenance, following a synchronised mating. From day 40 to day 90 (mid-pregnancy, MP), ewes were provided with either 80% (M) or 140% (H) of ME requirements. After 90 days of gestation, all ewes were fed to meet requirements for late pregnancy. During EP, mean live weight (LW) and body condition score (BCS) change of ewes were -6.3, -0.8 and +6.0kg and -0.02, +0.10 and +0.22 units in the L-, M- and H-EP treatments, respectively. During MP, mean LW and BCS change were -0.8 and +4.9kg and -0.09 and +0.09 units in the M- and H-MP treatments, respectively (P 0. 05) on conception rate, although there tended to be an inverse relationship (P = 0.085) between plane of nutrition in EP and plasma progesterone concentrations at day 42 of gestation. EP nutrition influenced foetal development with lambs from ewes offered diet L-EP being smaller (P

Molecular biology: the key to personalised treatment in radiation oncology?

We know considerably more about what makes cells and tissues resistant or sensitive to radiation than we did 20 years ago. Novel techniques in molecular biology have made a major contribution to our understanding at the level of signalling pathways. Before the ���New Biology��� era, radioresponsiveness was defined in terms of physiological parameters designated as the five Rs. These are: repair, repopulation, reassortment, reoxygenation and radiosensitivity. Of these, only the role of hypoxia proved to be a robust predictive and prognostic marker, but radiotherapy regimens were nonetheless modified in terms of dose per fraction, fraction size and overall time, in ways that persist in clinical practice today. The first molecular techniques were applied to radiobiology about two decades ago and soon revealed the existence of genes/proteins that respond to and influence the cellular outcome of irradiation. The subsequent development of screening techniques using microarray technology has since revealed that a very large number of genes fall into this category. We can now obtain an adequately robust molecular signature, predicting for a radioresponsive phenotype using gene expression and proteomic approaches. In parallel with these developments, functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) can now detect specific biological molecules such as haemoglobin and glucose, so revealing a 3D map of tumour blood flow and metabolism. The key to personalised radiotherapy will be to extend this capability to the proteins of the molecular signature that determine radiosensitivity.

Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs

Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2?kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of these were novel, and could be grouped into DNA families based upon sequence similarities. Significant inter-specific variation in abundance of cryptic repetitive DNA and DNA families was observed. Repbase scans show that a large proportion of cryptic repetitive DNA was identified as transposable elements (TEs). We argue that a large number of TEs and their transpositional activity may be linked to differential rates of DNA multiplication and recombination. This is likely to be an important factor explaining inter-specific variation in genome stability and hence microsatellite marker development success rates. Gastropods also differed significantly in the type of TEs classes (autonomous vs non-autonomous) observed. We propose that dissimilar transpositional mechanisms differentiate the TE classes in terms of their propensity for transposition, fixation and/or silencing. Consequently, the phylogenetic conservation of non-autonomous TEs, such as CvA, suggests that dispersal of these elements may have behaved as microsatellite-inducing elements. Results seem to indicate that, compared to autonomous, non-autonomous TEs maybe have a more active role in genome rearrangement processes. The implications of the findings for genomic rearrangement, stability and marker development are discussed.

miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro

The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself controlled extensively by a variety of genetic regulatory mechanisms. microRNA (miR), an abundant class of small non-coding RNA, are emerging as important post-transcriptional regulators of gene expression in the brain. Increasing evidence indicates that miR regulate both the development and function of the nervous system. Moreover, deficiency in miR function has also been implicated in a number of neurological disorders. Expression profile analysis of miR is necessary to understand their complex role in the regulation of gene expression during the development and differentiation of cells. Here we present a comparative study of miR expression profiles in neuroblastoma, in cortical development, and in neuronal differentiation of embryonic stem (ES) cells. By microarray profiling in combination with real time PCR we show that miR-7 and miR-214 are modulated in neuronal differentiation (as compared to miR-1, -16 and -133a), and control neurite outgrowth in vitro. These findings provide an important step toward further elucidation of miR function and miR-related gene regulatory networks in the mammalian central nervous system. (C) 2010 Elsevier Inc. All rights reserved.

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 K-m = 26.7 mu M, V-max = 0.43 mu M/mg protein/min; RIF-1 K-m = 33.5 mu M, V-max = 0.42 mu M/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 K-m = 37.5 mu M; V-max = 1.4 mu M/mg protein/min; RIF-1 K-m = 37.5 mu M; V-max = 1.2 mu M/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (K-m = 4 mu M, V-max = 3.5 pmol/mg protein/min) and normal kidney (K-m = 4 mu M, V-max = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Development of a novel DNA microarray to detect bacterial pathogens in patients with chronic obstructive pulmonary disease (COPD)

A novel microarray was constructed with DNA PCR product probes targeting species specific functional genes of nine clinically significant respiratory pathogens, including the Gram-positive organisms (Streptococcus pneumoniae, Streptococcus pyogenes), the Gram-negative organisms (Chlamydia pneumoniae, Coxiella burnetii Haemophilus spp., Legionella pneumophila, Moraxella catarrhalis, and Pseudomonas aeruginosa), as well as the atypical bacterium, Mycoplasma pneumoniae. In a "proof-of-concept" evaluation of the developed microarray, the microarray was compared with real-time PCR from 14 sputum specimens from COPD patients. All of the samples positive for bacterial species in real-time PCR were also positive for the same bacterial species using the microarray. This study shows that a microarray using PCR probes is a potentially useful method to monitor the populations of bacteria in respiratory specimens and can be tailored to specific clinical needs such as respiratory infections of particular patient populations, including patients with cystic fibrosis and bronchiectasis. (C) 2010 Elsevier B.V. All rights reserved.

Development and clinical validation of a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan�� based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.