Quantification of Hox and Surfactant Protein-B Transcription during Murine Lung Development.

Abstract BACKGROUND: Genetic processes underlying fetal lung development and maturation are incompletely understood. Better knowledge of these processes would provide insights into the causes of lung malformations and prevention of respiratory distress syndrome and the potential adverse effects of glucocorticoids. Hox genes are involved in the lung branching morphogenesis and maturation of respiratory epithelium, but their expression pattern remains to be defined. OBJECTIVES: We hypothesized that genes involved in lung branching would be downregulated during early development, whereas those involved in maturation would be unchanged or upregulated. METHODS: TaqMan real-time primers and probes were designed for all 39 murine Hox genes, and the murine SP-B gene and transcription profiles of these genes were obtained from whole lungs isolated at e14.5, e16.5, e18.5, e19.5 and postnatal days 1 and 20. RESULTS: Hox genes in clusters A and B, specifically those between paralog groups 3 and 7, were the most represented, with Hoxa4 and Hoxa5 being the most highly transcribed. A wave of reduced transcription in 16 Hox genes, coincident with increased SP-B transcription, was observed with advancing gestation. Consistently high transcription of Hoxa5 from e14.5 to postnatal day 20 may indicate that sustained transcription is required for normal lung maturation. When e15.5 lungs were cultured with dexamethasone, Hoxb6, Hoxb7 and Hoxb8 levels were significantly upregulated, creating the potential for modulation of diverse downstream target genes. CONCLUSIONS: Improved understanding of the genetic processes underlying lung development afforded by our Q-PCR platform may allow development of more specific methods for inducing fetal lung maturation.

Apoptosis is initiated in human keratinocytes exposed to signalling factors from microbeam irradiated cells.

PURPOSE: There is now no doubt that bystander signalling from irradiated cells occurs and causes a variety of responses in cells not targeted by the ionizing track. However, the mechanisms underlying these processes are unknown and the relevance to radiotherapy and risk assessment remains controversial. Previous research by our laboratory has shown bystander effects in a human keratinocyte cell line, HPV-G cells, exposed to medium from gamma irradiated HPV-G cells. The aim of this work was to investigate if similar mechanisms to those identified in medium transfer experiments occurred in these HPV-G cells when they are in the vicinity of microbeam irradiated cells. Demonstration of a commonality of mechanisms would support the idea that the process is not artifactual. MATERIALS AND METHODS: HPV-G cells were plated as two separate populations on mylar dishes. One population was directly irradiated using a charged particle microbeam (1 - 10 protons). The other population was not irradiated. Bystander factor-induced apoptosis was investigated in both populations following treatment by monitoring the levels of reactive oxygen species and mitochondrial membrane potential using fluorescent probes. Expression of the anti-apoptotic protein, bcl-2, and cytochrome c were determined, as well as apoptosis levels. RESULTS: Microbeam irradiation induced increases in reactive oxygen species and decreases in mitochondrial membrane potential at 6 h post-exposure, increased expression of bcl-2 and cytochrome c release at 6.5 h and increased apoptosis at 24 h. CONCLUSION: This study shows that similar bystander signalling pathways leading to apoptosis are induced following microbeam irradiation and following medium transfer. This demonstrates that the mechanisms involved are common across different radiation qualities and conditions and indicates that they may be relevant in vivo.

Open-ended microwave oven for flip-chip assembly

A novel open-ended waveguide cavity resonator for the microwave curing of bumps, underfills and encapsulants is described. The open oven has the potential to provide fast alignment of devices during flip-chip assembly, direct chip attach, surface mount assembly or wafer-scale level packaging. The prototype microwave oven was designed to operate at X-band for ease of testing, although a higher frequency version is planned. The device described in the paper takes the form of a waveguide cavity resonator. It is approximately square in cross-section and is filled with a low-loss dielectric with a relative permittivity of 6. It is excited by end-fed probes in order to couple power preferentially into the TM3,3,k mode with the object of forming nine 'hot-spots' in the open end. Low power tests using heat sensitive film demonstrate clearly that selective heating in multiple locations in the open end of the oven is achievable.

Gender differences, responsiveness and memory of a potentially painful event in hermit crabs

Nonreflexive responses to a noxious event and prolonged memory are key criteria of a pain experience. In a previous study, hermit crabs, Pagurus bernhardus, that received a small electric shock within their shell often temporarily evacuated the shell and some groomed their abdomen and/or moved away from their vital resource. Most, however, returned to the shell. When offered a new shell 20 s later, shocked crabs were more likely than nonshocked crabs to approach and move into a new shell and did so more quickly (Elwood & Appel 2009, Animal Behaviour, 77, 1243-1246). Here we examined how increasing the time between the shock and the offering of a new shell influences the response. There was evidence of a memory of the aversive shock that lasted at least 1 day. Crabs tested after 30 min and 1 day were more likely to approach the shell and new shells were more likely to be taken 30 min after the shock. Shocked crabs approached the new shell more quickly and used fewer probes of the chelipeds prior to moving in and these results were stable over time and significant for specific times up to 1 day. Females were more likely than males to evacuate shells and did so after fewer shocks. These results extend previous work and demonstrate an extended memory of having been shocked. The findings are consistent with respect to criteria for pain that are accepted for vertebrates.

Pain experience in hermit crabs?

Pain may be inferred when the responses to a noxious stimulus are not reflexive but are traded off against other motivational requirements, the experience is remembered and the situation is avoided in the future. To investigate whether decapods feel pain we gave hermit crabs, Pagurus bernhardus, small electric shocks within their shells. Only crabs given shocks evacuated their shells indicating the aversive nature of the stimulus, but fewer crabs evacuated from a preferred species of shell indicating a motivational trade-off. Some crabs that evacuated attacked the shell in the manner seen in a shell fight. Most crabs, however, did not evacuate at the stimulus level we used, but when these were subsequently offered a new shell, shocked crabs were more likely to approach and enter the new shell. Furthermore, they approached that shell more quickly, investigated it for a shorter time and used fewer cheliped probes within the aperture prior to moving in. Thus the experience of the shock altered future behaviour in a manner consistent with a marked shift in motivation to get a new shell to replace the one occupied. The results are consistent with the idea of pain in these animals. (C) 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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.

Methods for measuring proteasome activity: Current limitations and future developments

The proteasome has been validated as a therapeutic target, with proteasome inhibitors showing particular efficacy in the treatment of Multiple Myeloma. A wide range of methods have been developed to profile proteasome activity. These include the current method of choice fluorogenic peptide substrates, as well as bioluminescent imaging, immunological methods, and more recently, site-specific fluorescent probes. The aim of this review is to evaluate the currently available methods for profiling proteasome activity and their suitability for use in translational studies. Ongoing development of techniques for profiling proteasome activity will facilitate future research into proteasome-related pathologies, thus accelerating the development of more specific drug regimes. (C) 2010 Elsevier Ltd. All rights reserved.

Impact of Sample Heterogeneity on Methylation Analysis

The recent emergence of high-throughput arrays for methylation analysis has made the influence of tumor content on the interpretation of methylation levels increasingly pertinent. However, to what degree does tumor content have an influence, and what degree of tumor content makes a specimen acceptable for accurate analysis remains unclear. Taking a systematic approach, we analyzed 98 unselected formalin-fixed and paraffin-embedded gastric tumors and matched normal tissue samples using the Illumina GoldenGate methylation assay. Unsupervised hierarchical clustering showed 2 separate clusters with a significant difference in average tumor content levels. The probes identified to be significantly differentially methylated between the tumors and normals also differed according to the tumor content of the samples included, with the sensitivity of identifying the

Development and clinical validation of multiplex TaqMan® assays for rapid diagnosis of viral gastroenteritis.

There is a need to provide rapid, sensitive, and often high throughput detection of pathogens in diagnostic virology. Viral gastroenteritis is a serious health issue often leading to hospitalization in the young, the immunocompromised and the elderly. The common causes of viral gastroenteritis include rotavirus, norovirus (genogroups I and II), astrovirus, and group F adenoviruses (serotypes 40 and 41). This article describes the work-up of two internally controlled multiplex, probe-based PCR assays and reports on the clinical validation over a 3-year period, March 2007 to February 2010. Multiplex assays were developed using a combination of TaqMan™ and minor groove binder (MGB™) hydrolysis probes. The assays were validated using a panel of 137 specimens, previously positive via a nested gel-based assay. The assays had improved sensitivity for adenovirus, rotavirus, and norovirus (97.3% vs. 86.1%, 100% vs. 87.8%, and 95.1% vs. 79.5%, respectively) and also more specific for targets adenovirus, rotavirus, and norovirus (99% vs. 95.2%, 100% vs. 93.6%, and 97.9% vs. 92.3%, respectively). For the specimens tested, both assays had equal sensitivity and specificity for astrovirus (100%). Overall the probe-based assays detected 16 more positive specimens than the nested gel-based assay. Post-introduction to the routine diagnostic service, a total of 9,846 specimens were processed with multiplex 1 and 2 (7,053 pediatric, 2,793 adult) over the 3-year study period. This clinically validated, probe-based multiplex testing algorithm allows highly sensitive and timely diagnosis of the four most prominent causes of viral gastroenteritis.

On the self-aggregation and fluorescence quenching aptitude of surfactant ionic liquids

The aggregation behavior in aqueous solution of a number of ionic liquids was investigated at ambient conditions by using three techniques: fluorescence, interfacial tension, and H-1 NMR spectroscopy. For the first time, the fluorescence quenching effect has been used for the determination of critical micelle concentrations. This study focuses on the following ionic liquids: [C(n)mpy]Cl (1-alkyl-3-methylpyridinium chlorides) with different linear alkyl chain lengths (n = 4, 10, 12, 14, 16, or 18), [C(12)mpip]Br (1-dodecyl-1-methylpiperidinium bromide), [C(12)mpy]Br (1-dodecyl-3-methylpyridinium bromide), and [C(12)mpyrr]Br (1-dodecyl-1-methylpyrrolidinium bromide). Both the influence of the alkyl side-chain length and the type of ring in the cation (head) on the CMC were investigated. A comparison of the self-aggregation behavior of ionic liquids based on 1-alkyl-3-methylpyridinium and 1-alkyl-3-methylpyridinium cations is provided. It was observed that 1-alkyl-3-methylpyridinium ionic liquids could be used as quenchers for some fluorescence probes (fluorophores). As a consequence, a simple and convenient method to probe early evidence of aggregate formation was established.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 May 2009-31 July 2009

This article documents the addition of 512 microsatellite marker loci and nine pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Alcippe morrisonia morrisonia, Bashania fangiana, Bashania fargesii, Chaetodon vagabundus, Colletes floralis, Coluber constrictor flaviventris, Coptotermes gestroi, Crotophaga major, Cyprinella lutrensis, Danaus plexippus, Fagus grandifolia, Falco tinnunculus, Fletcherimyia fletcheri, Hydrilla verticillata, Laterallus jamaicensis coturniculus, Leavenworthia alabamica, Marmosops incanus, Miichthys miiuy, Nasua nasua, Noturus exilis, Odontesthes bonariensis, Quadrula fragosa, Pinctada maxima, Pseudaletia separata, Pseudoperonospora cubensis, Podocarpus elatus, Portunus trituberculatus, Rhagoletis cerasi, Rhinella schneideri, Sarracenia alata, Skeletonema marinoi, Sminthurus viridis, Syngnathus abaster, Uroteuthis (Photololigo) chinensis, Verticillium dahliae, Wasmannia auropunctata, and Zygochlamys patagonica. These loci were cross-tested on the following species: Chaetodon baronessa, Falco columbarius, Falco eleonorae, Falco naumanni, Falco peregrinus, Falco subbuteo, Didelphis aurita, Gracilinanus microtarsus, Marmosops paulensis, Monodelphis Americana, Odontesthes hatcheri, Podocarpus grayi, Podocarpus lawrencei, Podocarpus smithii, Portunus pelagicus, Syngnathus acus, Syngnathus typhle,Uroteuthis (Photololigo) edulis, Uroteuthis (Photololigo) duvauceli and Verticillium albo-atrum. This article also documents the addition of nine sequencing primer pairs and sixteen allele specific primers or probes for Oncorhynchus mykiss and Oncorhynchus tshawytscha; these primers and assays were cross-tested in both species.

Development of an allele-specific real-time PCR assay for discrimination and quantification of p63 R279H mutation in EEC syndrome

The ectrodactyly-ectodermal dysplasiaclefting syndrome is a rare autosomal dominant disorder caused by heterozygous mutations in the p63 gene, a transcription factor belonging to the p53 family. The majority of cases of ectrodactyly-ectodermal dysplasia syndrome are caused by de novo mutations and are therefore sporadic in approximately 60% of patients. The substitution of arginine to histidine (R279H), due to a c.836G>A mutation in exon 7 of the p63 gene, represents 55% of the identified mutations and is considered a mutational hot spot. A quantitative and sensitive real-time PCR was performed to quantify both wild-type and R279H alleles in DNA extracted from peripheral blood and RNA from cultured epithelial cells. Standard curves were constructed for both wild-type and mutant probes. The sensitivity of the assay was determined by generating serial dilutions of the DNA isolated from heterozygous patients (50% of alleles mutated) with wild-type DNA, thus obtaining decreasing percentages of p63 R279H mutant allele (50%, 37.5%, 25%, 12.5%, 10%, 7.5%, 5%, 2.5%, and 0.0%). The assay detected up to 1% of the mutant p63. The high sensitivity of the assay is of particular relevance to prenatal diagnosis and counseling and to detect therapeutic effects of drug treatment or gene therapy aimed at reducing the amount of mutated p63. © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Structural mimics for the active site of [NiFe] hydrogenase

Hydrogenases are enzymes that catalyse the reversible two-electron oxidation of H-2. The [NiFe] hydrogenases have been characterised spectroscopically and by single crystal X-ray diffraction, and show an active site incorporating a heterobinuclear [NiFe] centre bridged by two cysteine S-donors. Low molecular weight synthetic complex models, which structurally mimic the dithiolate-bridged [NiFe] centre, serve as important probes of structure and chemistry at the active site and are the subject of this review. (C) 2001 Elsevier Science B.V. All rights reserved.

Early-type stars observed in the ESO UVES Paranal Observatory Project - III. Sub-parsec and AU-scale structure in the interstellar medium

UVES interstellar observations from the Paranal Observatory Project are presented for early-type stars located in the line of sight to the nearby open clusters IC 2391 (Omni Vel) and NGC 6475 (M7), with spectroscopic resolution R similar to 80 000 and signal-to-noise ratios in the Ti II (3383 angstrom), Ca II K, CH+ (4232 angstrom), Na I D and K I (7698 angstrom) lines of several hundred. The sightlines are a mixture of cluster and non-cluster objects. A total of 22 early-type stars (A and B type) are present in our sample towards IC 2391, with 21 towards NGC 6475/M7, and enable us to probe for differences in column density on scales from similar to 0.07 to 7.3 and similar to 0.05 to 4.9 pc in the respective clusters. Additionally, towards Praesepe the Na I D interstellar variation only is probed towards 13 sightlines and transverse scales of similar to 0.16-10.7 pc at R = 70 000. Towards IC 2391 variations are found in Ti II, Ca II K and Na I D column density in different sightlines of up to 0.7, 1.0 and 1.8 dex (excluding one star), respectively. This kind of variability correlates well with the Hipparcos parallax of the objects, and probes structure within the Local Bubble. For cluster-only objects the variations are 0.3, 0.3 and 0.5 dex, respectively. For the field of view towards NGC6475 the corresponding maximum variations are somewhat smaller, being 0.5, 0.3, 0.8 and 1.0 dex for Ti II, Ca II K, Na I and K I, respectively, for all objects and 0.4, 0.2, 0.6 and 0.7 dex for the cluster-only objects. These are uncorrelated with parallax, and again demonstrate that Ca II K tends to be more smoothly distributed than Na I D. A few likely cluster sightlines show evidence for CH+ and variations in this molecular species of a factor of 10 in equivalent width over sub-pc scales. Towards Praesepe variation in interstellar Na I D is small, being a maximum of only similar to 0.4 dex (including measurement errors), but with fewer sightlines studied. Overall, the scatter in the data is similar for the singly ionized species Ti II and Ca II, lending more support to the hypothesis that these two species sample similar parts of the interstellar medium (ISM). This also appears to be the case for the neutral species Na I D and K I in the one cluster studied. Finally, multiple-epoch observations from a variety of archive sources are used to search for astronomical unit (au) scale structure in the ISM towards 46 sightlines. There are tentative indications of structure on scales of tens to thousands of au for three sightlines. Future observations will confirm the veracity or otherwise of the time-variable components and others presented.

Larval Transport Modeling of Deep-Sea Invertebrates Can Aid the Search for Undiscovered Populations

Background: Many deep-sea benthic animals occur in patchy distributions separated by thousands of kilometres, yet because deep-sea habitats are remote, little is known about their larval dispersal. Our novel method simulates dispersal by combining data from the Argo array of autonomous oceanographic probes, deep-sea ecological surveys, and comparative invertebrate physiology. The predicted particle tracks allow quantitative, testable predictions about the dispersal of benthic invertebrate larvae in the south-west Pacific. Principal Findings: In a test case presented here, using non-feeding, non-swimming (lecithotrophic trochophore) larvae of polyplacophoran molluscs (chitons), we show that the likely dispersal pathways in a single generation are significantly shorter than the distances between the three known population centres in our study region. The large-scale density of chiton populations throughout our study region is potentially much greater than present survey data suggest, with intermediate ‘stepping stone’ populations yet to be discovered. Conclusions/Significance: We present a new method that is broadly applicable to studies of the dispersal of deep-sea organisms. This test case demonstrates the power and potential applications of our new method, in generating quantitative, testable hypotheses at multiple levels to solve the mismatch between observed and expected distributions: probabilistic predictions of locations of intermediate populations, potential alternative dispersal mechanisms, and expected population genetic structure. The global Argo data have never previously been used to address benthic biology, and our method can be applied to any non-swimming larvae of the deep-sea, giving information upon dispersal corridors and population densities in habitats that remain intrinsically difficult to assess.