Extreme-ultraviolet refractive optics.

Refraction is a well-known optical phenomenon that alters the direction of light waves propagating through matter. Microscopes, lenses and prisms based on refraction are indispensable tools for controlling light beams at visible, infrared, ultraviolet and X-ray wavelengths1. In the past few decades, a range of extreme-ultraviolet and soft-X-ray sources has been developed in laboratory environments2-4 and at large-scale facilities5,6. But the strong absorption of extreme-ultraviolet radiation in matter hinders the development of refractive lenses and prisms in this spectral region, for which reflective mirrors and diffractive Fresnel zone plates7 are instead used for focusing. Here we demonstrate control over the refraction of extreme-ultraviolet radiation by using a gas jet with a density gradient across the profile of the extreme-ultraviolet beam. We produce a gas-phase prism that leads to a frequency-dependent deflection of the beam. The strong deflection near to atomic resonances is further used to develop a deformable refractive lens for extreme-ultraviolet radiation, with low absorption and a focal length that can be tuned by varying the gas pressure. Our results open up a route towards the transfer of refraction-based techniques, which are well established in other spectral regions, to the extreme-ultraviolet domain.

Time-Resolved Measurement of Interatomic Coulombic Decay Induced by Two-Photon Double Excitation of Ne_{2}.

The hitherto unexplored two-photon doubly excited states [Ne^{*}(2p^{-1}3s)]_{2} were experimentally identified using the seeded, fully coherent, intense extreme ultraviolet free-electron laser FERMI. These states undergo ultrafast interatomic Coulombic decay (ICD), which predominantly produces singly ionized dimers. In order to obtain the rate of ICD, the resulting yield of Ne_{2}^{+} ions was recorded as a function of delay between the extreme ultraviolet pump and UV probe laser pulses. The extracted lifetimes of the long-lived doubly excited states, 390(-130/+450) fs, and of the short-lived ones, less than 150 fs, are in good agreement with ab initio quantum mechanical calculations.

IRF6 mutation screening in non-syndromic orofacial clefting: analysis of 1521 families.

Van der Woude syndrome (VWS) is an autosomal dominant malformation syndrome characterized by orofacial clefting (OFC) and lower lip pits. The clinical presentation of VWS is variable and can present as an isolated OFC, making it difficult to distinguish VWS cases from individuals with non-syndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with non-syndromic OFCs. Screening for IRF6 mutations in apparently non-syndromic cases has been performed in several modestly sized cohorts with mixed results. In this study, we screened 1521 trios with presumed non-syndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and non-syndromic OFCs. We combined our results with other similar studies (totaling 2472 families) and conclude that causal IRF6 mutations are found in 0.24-0.44% of apparently non-syndromic OFC families. We suggest that clinical mutation screening for IRF6 be considered for certain family patterns such as families with mixed types of OFCs and/or autosomal dominant transmission.

Slow Interatomic Coulombic Decay of Multiply Excited Neon Clusters.

Ne clusters (∼5000 atoms) were resonantly excited (2p→3s) by intense free electron laser (FEL) radiation at FERMI. Such multiply excited clusters can decay nonradiatively via energy exchange between at least two neighboring excited atoms. Benefiting from the precise tunability and narrow bandwidth of seeded FEL radiation, specific sites of the Ne clusters were probed. We found that the relaxation of cluster surface atoms proceeds via a sequence of interatomic or intermolecular Coulombic decay (ICD) processes while ICD of bulk atoms is additionally affected by the surrounding excited medium via inelastic electron scattering. For both cases, cluster excitations relax to atomic states prior to ICD, showing that this kind of ICD is rather slow (picosecond range). Controlling the average number of excitations per cluster via the FEL intensity allows a coarse tuning of the ICD rate.

Efficient autoionization following intense laser-cluster interactions.

Electron emission as a result of the interaction of clusters with intense laser pulses is commonly understood in terms of direct and evaporative ionization processes. In contrast, we provide evidence here of an important role played by autoionization in intense field ionization of molecular oxygen clusters. Superexcited states are populated during the cluster expansion, and their autoionization is observed on a ns time scale. Decay processes on fs to ps time scales are obscured by energy exchange of the emitted electrons with the environment.

Rare-gas clusters in intense extreme-ultraviolet pulses from a high-order harmonic source.

We report evidence for two previously unidentified effects in the ionization of rare-gas clusters by intense extreme-ultraviolet pulses. First, electron spectra indicate multistep photoemission with increasing isotropy for larger clusters due to electron-atom collisions. Second, very slow (meV) electrons are interpreted as the first experimental evidence for Rydberg-like atomic state formation in the nanoplasma expansion. Only small fractions of Xe2+ ions were found, in sharp contrast to previous results recorded under comparable conditions [Murphy et al., Phys. Rev. Lett. 101, 203401 (2008).

Tracing electron-ion recombination in nanoplasmas produced by extreme-ultraviolet irradiation of rare-gas clusters.

We investigate electron-ion recombination in nanoplasmas produced by the ionization of rare-gas clusters with intense femtosecond extreme-ultraviolet (XUV) pulses. The relaxation dynamics following XUV irradiation is studied using time-delayed 790-nm pulses, revealing the generation of a large number of excited atoms resulting from electron-ion recombination. In medium-sized Ar-Xe clusters, these atoms are preferentially created in the Xe core within 10 ps after the cluster ionization. The ionization of excited atoms serves as a sensitive probe for monitoring the cluster expansion dynamics up to the ns time scale.

TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.

There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.

Evidence for chirped Auger-electron emission.

Auger decay carries valuable information about the electronic structure and dynamics of atoms, molecules, and solids. Here we furnish evidence that under certain conditions Auger electrons are subject to an energetic chirp. The effect is disclosed in time-resolved streaking experiments on the Xe NOO and Kr MNN Auger decay using extreme-ultraviolet pulses from the free-electron laser in Hamburg as well as from a high-order harmonic laser source. The origin of this effect is found to be an exchange of energy between the Auger electron and an earlier emitted correlated photoelectron. The observed time-dependent spectral modulations are understood within an analytical model and confirmed by extensive computer simulations.

The caspase-9 derived C-terminal fragment of cytokeratin 18 modulates topoisomerase action.

During early apoptosis the 33 amino acid C-terminal cytokeratin 18 (CK18) fragment is released by caspase-9 cleavage at the 393DALD/S site. This basic peptide relocates from the cytoskeleton to the nucleoplasm as shown by confocal laser scanning. It is shown that the C-terminal peptide modulates topoisomerase activity as measured by relaxation of plasmid DNA. In an in vitro assay recombinant caspase-induced chromatin condensation is inhibited by the peptide and at the electron microscopical level a clear inhibition of nucleolar breakdown was observed in its presence. We hypothesize that the C-terminal CK18 fragment exerts an effect in the nucleolus by stimulating rRNA transcription and processing via modulation of enzymatic activity of topoisomerase I. This leads to preservation of general transcriptional activity required to exert active steps during early stages of programmed cell death.

Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding.

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridisation.

AIM AND METHOD: We analysed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridisation (array-CGH). RESULTS: Of 83 syndromic cases analysed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in five of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 non-syndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has a cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software. CONCLUSION: We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.

Poorer outcome in stromal HIF-2 alpha- and CA9-positive colorectal adenocarcinomas is associated with wild-type TP53 but not with BNIP3 promoter hypermethylation or apoptosis.

Stromal expression of hypoxia inducible factor 2 alpha (HIF-2 alpha) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2 alpha- and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5-8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2 alpha, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2 alpha and CA9 stromal-positive CRCs was associated with wild-type TP53 (P=0.001 and P=0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P=0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis. These results suggest that the biology of CRC cells can be modified by alterations in the tumour microenvironment under conditions of tumour hypoxia.

Short-term results of our first 49 Scandanavian total ankle replacements (STAR).

BACKGROUND: Forty-seven consecutive patients treated for ankle arthritis with a Scandinavian total ankle replacement (STAR) by one surgeon were investigated retrospectively. MATERIALS AND METHODS: A modification of the Foot Function Index (FFI), which scores pain and task difficulties, was followed prospectively. Patients were assessed clinically and radiologically. Failure was defined as revision of the prosthesis or arthrodesis for any reason. RESULTS: In 47 patients (16 male, 31 female) 49 total ankle replacements were carried out between May 1999 and June 2004. Indication for surgery was end stage arthritis for rheumatoid arthritis in 29 cases, post-traumatic arthritis in 12, osteoarthritis in five and arthritis secondary to degenerative flatfoot in three. Mean followup time was 28 (12 to 67) months. The modified FFI (range, 0 to 100, a high score meaning more pain and disability) improved significantly from 59 before to 35 after surgery. The mean postoperative Kofoed ankle score was 68. Sixteen procedures were complicated by fractures or temporary neurological damage. At the time of followup, 45 prostheses survived, while four replacements had failed. Radiological examination at followup showed radiolucent lines, osteolysis, and malposition of the components in 31 cases. CONCLUSION: Our results are comparable with those reported in the literature. The clinical outcome improved significantly. Due to aseptic and septic loosening, 8.2% of the prosthesis failed.

Defective expression of p56lck in an infant with severe combined immunodeficiency.

Severe combined immune deficiency (SCID) is a heterogeneous disorder characterized by profound defects in cellular and humoral immunity. We report here an infant with clinical and laboratory features of SCID and selective CD4 lymphopenia and lack of CD28 expression on CD8(+) T cells. T cells from this patient showed poor blastogenic responses to various mitogens and IL-2. Other T cell antigen receptor- induced responses, including upregulation of CD69, were similarly inhibited. However, more proximal T cell antigen receptor signaling events, such as anti-CD3 induced protein tyrosine phosphorylation, phosphorylation of mitogen-associated protein kinase, and calcium mobilization were intact. Although p59fyn and ZAP-70 protein tyrosine kinases were expressed at normal levels, a marked decrease in the level of p56lck was noted. Furthermore, this decrease was associated with the presence of an alternatively spliced lck transcript lacking the exon 7 kinase encoding domain. These data suggest that a deficiency in p56lck expression can produce a SCID phenotype in humans.

IRF6 and SPRY4 Signaling Interact in Periderm Development.

Rare mutations in IRF6 and GRHL3 cause Van der Woude syndrome, an autosomal dominant orofacial clefting disorder. Common variants in IRF6 and GRHL3 also contribute risk for isolated orofacial clefting. Similarly, variants within genes that encode receptor tyrosine kinase (RTK) signaling components, including members of the FGF pathway, EPHA3 and SPRY2, also contribute risk for isolated orofacial clefting. In the mouse, loss of Irf6 or perturbation of Fgf signaling leads to abnormal oral epithelial adhesions and cleft palate. Oral adhesions can result from a disruption of periderm formation. Here, we find that IRF6 and SPRY4 signaling interact in periderm function. We crossed Irf6 heterozygous ( Irf6+/-) mice with transgenic mice that express Spry4 in the basal epithelial layer ( TgKRT14::Spry4). While embryos with either of these mutations can have abnormal oral adhesions, using a new quantitative assay, we observed a nonadditive effect of abnormal oral epithelial adhesions in the most severely affected double mutant embryos ( Irf6+/-;TgKRT14::Spry4). At the molecular level, the sites of abnormal oral adhesions maintained periderm-like cells that express keratin 6, but we observed abnormal expression of GRHL3. Together, these data suggest that Irf6 and RTK signaling interact in regulating periderm differentiation and function, as well as provide a rationale to screen for epistatic interactions between variants in IRF6 and RTK signaling pathway genes in human orofacial clefting populations.

Comparison of phenotypic and genotypic features in human primary large bowel carcinomas and lymph node metastases.

In order to test the contention that metastasis is a selective process and that therefore metastases might show a more restricted pattern of phenotypic and genotypic characteristics than primary tumors, we compared the expression of carcinoembryonic antigen, Ca 19-9, secretory component, serotonin, and mucin production as well as flow cytometric data on DNA content and percentage of S-phase cells in 87 primary large bowel carcinomas and their lymph node metastases. In a majority of the cases primary tumors and their metastases were largely identical with regard to the examined phenotypic features. In discrepant cases, however, metastases did not invariably show a more restricted pattern than primary tumors, indicating high differentiational plasticity of primary and metastatic colorectal cancer cells. In contrast, in a number of cases genotypic discrepancies were observed. We conclude that phenotypic characteristics of colorectal cancer cells cannot be used to study the pathogenesis of lymph node metastasis. Genotypic studies, however, suggest that lymphogenic metastasis may be a selective event.

Retrospective analysis of the prognostic significance of DNA content and proliferative activity in large bowel carcinoma.

In the present study we have evaluated the prognostic significance of ploidy levels and proliferative activity in 279 cases of large bowel carcinomas which were included in a surgical prospective randomized trial. Ploidy levels and proliferative activity were determined on nuclei isolated from paraffin-embedded tissues of 279 colorectal carcinoma patients, with a mean follow-up of 51.9 months. Product limit survival analysis demonstrated a borderline significant association (P = 0.0689 by generalized Breslow; P = 0.0336 by generalized Savage) between ploidy and survival, with a 75th quantile survival of 49.8 months for patients with diploid tumors and 35.9 months for patients with aneuploid tumors. After stratification for staging, Dukes' C cases showed a statistically significant association between tumor ploidy and survival (P = 0.0224 by generalized Breslow, P = 0.0110 by generalized Savage). Product limit survival analysis for proliferative activity and survival showed a similar outcome with the strongest association in Dukes's C stage of disease (75th quantile survival of 38.9 months for low proliferative and 18.0 months for high proliferative tumors).

Full Spectrum of Postnatal Tooth Phenotypes in a Novel Irf6 Cleft Lip Model.

Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities, including hypodontia and supernumerary teeth, aberrant crown and root morphologies, and enamel defects, although the underlying mechanistic link is poorly understood. As most CLP genes are expressed throughout the oral epithelium, the authors hypothesized that the expression of CLP genes may persist in the dental epithelium and thus, in addition to their earlier role in labiopalatine development, may play an important functional role in subsequent tooth patterning and amelogenesis. To address this, the authors generated a unique conditional knockout model involving the major CLP gene, Irf6, that overcomes the previously reported perinatal lethality to enable assessment of any posteruption dental phenotypes. A dental epithelium-specific Irf6 conditional knockout (Irf6-cKO) mouse was generated via a Pitx2-Cre driver line. Dental development was analyzed by microcomputed tomography, scanning electron microscopy, histology, immunohistochemistry, and quantitative polymerase chain reaction. Irf6-cKO mice displayed variable hypodontia, occasional supernumerary incisors and molars, as well as crown and root patterning anomalies, including peg-shaped first molars and taurodontic and C-shaped mandibular second molars. Enamel density was reduced in preeruption Irf6-cKO mice, and some shearing of enamel rods was noted in posteruption incisors. There was also rapid attrition of Irf6-cKO molars following eruption. Histologically, Irf6-cKO ameloblasts exhibited disturbances in adhesion and polarity, and delayed enamel formation was confirmed immunohistochemically. Altered structure of Hertwig's epithelial root sheath was also observed. These data support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. This data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual.

A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer.

The ETS family of genes are implicated in cancers such as Ewings sarcoma, acute myeloid leukemia and chronic myelomonocytic leukemia. Further, they have important functions in embryonic development. Hence, identification and characterization of members of this family are important. We identify a novel ETS family member, ELF3, and report its human and murine cDNA sequences. The mouse cDNA has an alternatively spliced transcript with an extra 60 bp inserted. Hence we present the organization of the murine Elf3 gene together with its exon/intron structure. This gene consists of 9 exons and 8 introns spanning 4.8 kb. ELF3 binds and transactivates ETS sequences and interestingly also shows the ability to bind a GGAT-like purine core, a preferential ETS1/ETS2 type binding site. The expression of ELF3, unlike most other ETS family members, is absent in hematopoietic cells and hematopoietic organs in humans and mice. Intriguingly, the gene is specifically expressed in cell lines of epithelial origin and in organs such as lung, stomach, intestine, kidney that have specialized epithelial cells. We localize the human gene to 1q32.2, a region that is amplified in epithelial tumors of the breast, lung and prostate. Finally, we show that ELF3 expression is increased in a lung carcinoma and adenocarcinoma, as compared to normal tissue. ELF3 is also expressed in cell lines derived from lung cancers. These results suggest that this novel ETS gene may be involved in lung tumorigenesis.