Selective Imaging of Damaged Bone Structure (Microcracks) Using a Targeting Supramolecular Eu(III) Complex As a Lanthanide Luminescent Contrast Agent

The synthesis and photophysical evaluation of a new supramolecular lanthanide complex is described which was developed as a luminescent contrast agent for bone structure analysis. We show that the Eu(III) emission of this complex is not pH dependent within the physiological pH range, and that its steady state emission is not significantly modulated by a series of group I and II as well as d-metal ions, and that this agent can be successfully employed to image mechanically formed cracks (scratches) in bone samples after 4 or 24 hours, using confocal laser-scanning microscopy.

Effect of signal intensity normalization on the multivariate analysis of spectral data in complex 'real-world' datasets

Spectral signal intensities, especially in 'real-world' applications with nonstandardized sample presentation due to uncontrolled variables/factors, commonly require additional spectral processing to normalize signal intensity in an effective way. In this study, we have demonstrated the complexity of choosing a normalization routine in the presence of multiple spectrally distinct constituents by probing a dataset of Raman spectra. Variation in absolute signal intensity (90.1% of total variance) of the Raman spectra of these complex biological samples swamps the variation in useful signals (9.4% of total variance), degrading its diagnostic and evaluative potential.

FBXL21 Association With Schizophrenia in Irish Family and Case-Control Samples

FBXL21 gene encodes an F-box containing protein functioning in the SCIP ubiquitin ligase complex. The role of the F-box protein is to recruit proteins designated for degradation to the ligase complex so they would be ubiquitinated. Using both family and case-control samples, we found consistent associations in and around FBXL21 gene. In the family sample (Irish study of high density schizophrenia families, ISHDSF, 1,350 subjects from 273 families), a minimal PDT P-value of 0.0011 was observed at rs31555. In the case-control sample (Irish case-control study of schizophrenia, ICCSS, 814 cases and 625 controls), significant associations were observed at two markers (rs1859427 P=0.0197, and rs6861170 P=0.0197). In haplotype analyses, haplotype 1-1 (C-T) of rs1859427-rs6861170 was overtransmitted in the ISHDSF (P=0.0437) and was over-represented in the ICCSS (P=0.0177). For both samples, the associated alleles and haplotypes were identical. These data suggested that FBXL21 maybe associated with schizophrenia in the Irish samples. (C) 2008 Wiley-Liss, Inc.

Analysis of luminescent samples using subtracted shifted Raman spectroscopy

A novel method of obtaining high-quality Raman spectra of luminescent samples was tested using cyclohexane solutions which had been treated with a fluorescent dye. The method involves removing the fixed pattern irregularity found in the spectra taken with CCD detectors by subtracting spectra taken at several different, closely spaced spectrometer positions. It is conceptually similar to SERDS (shifted excitation Raman difference spectroscopy) but has the distinct experimental advantage that it does not require a tunable laser source. The subtracted spectra obtained as the raw data are converted into a more recognisable and conventional form by iterative fitting of appropriate double Lorentzian functions whose peak parameters are then used to 'reconstruct' a conventional representation of the spectrum. Importantly, it is shown that the degree of uncertainty in the resultant 'reconstructed' spectra can be gauged reliably by comparing reconstructed spectra obtained at two different spectrometer shifts (delta and 2 delta), The method was illustrated and validated using a solvent (cyclohexane) the spectrum of which is well known and which contains both regions with complex overlapping bands and regions with isolated bands, Possible sources of error are discussed and it is shown that, provided the degree of uncertainty in the data is correctly characterised, it is completely valid to draw conclusions about the spectra of the sample on the basis of the reconstructed data. The acronym SSRS (subtracted shifted Raman spectroscopy; pronounced scissors) is proposed for this method, to distinguish it from the SERDS technique.

A MOLECULAR AND MORPHOLOGICAL ANALYSIS OF THE GYMNOGONGRUS-DEVONIENSIS (RHODOPHYTA) COMPLEX IN THE NORTH-ATLANTIC

The Gymnogongrus devoniensis (Greville) Schotter complex in the North Atlantic Ocean was elucidated by comparative molecular, morphological, and culture studies. Restriction fragment length patterns and hybridization data on organellar DNA revealed two distinct taxa in samples from Europe and eastern Canada. Nucleotide sequences for the intergenic spacer between the large and small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and the adjoining regions of both genes, differed by 12.5-13.4% between the two taxa. One of the taxa, which included material from the type locality of G. devoniensis at Torbay, Devon, England, was taken to represent authentic G. devoniensis. Within this taxon, samples from Ireland, England, northern France, northern Spain, and southern Portugal showed great morphological variation, particularly in habit, but their Rubisco spacer sequences were identical or differed by only a single nucleotide. Constant morphological features included the development, from a single auxiliary cell, of the spherical cystocarp with a thick mucilage sheath that appears to be typical of Gymnogongrus species with internal cystocarps. Two life-history types were found. Northern isolates underwent a direct-type life history, recycling apomictic females by carpospores, whereas the Portuguese isolate followed a heteromorphic life history in which carpospores gave rise to a crustose tetrasporophyte.

Genome-wide Association Study Implicates HLA-C*01:02 as a Risk Factor at the Major Histocompatibility Complex Locus in Schizophrenia

BACKGROUND: We performed a genome-wide association study (GWAS) to identify common risk variants for schizophrenia. METHODS: The discovery scan included 1606 patients and 1794 controls from Ireland, using 6,212,339 directly genotyped or imputed single nucleotide polymorphisms (SNPs). A subset of this sample (270 cases and 860 controls) was subsequently included in the Psychiatric GWAS Consortium-schizophrenia GWAS meta-analysis. RESULTS: One hundred eight SNPs were taken forward for replication in an independent sample of 13,195 cases and 31,021 control subjects. The most significant associations in discovery, corrected for genomic inflation, were (rs204999, p combined = 1.34 × 10(-9) and in combined samples (rs2523722 p combined = 2.88 × 10(-16)) mapped to the major histocompatibility complex (MHC) region. We imputed classical human leukocyte antigen (HLA) alleles at the locus; the most significant finding was with HLA-C*01:02. This association was distinct from the top SNP signal. The HLA alleles DRB1*03:01 and B*08:01 were protective, replicating a previous study. CONCLUSIONS: This study provides further support for involvement of MHC class I molecules in schizophrenia. We found evidence of association with previously reported risk alleles at the TCF4, VRK2, and ZNF804A loci.

Can arsenic-phytochelatin complex formation be used as an indicator for toxicity in Helianthus annuus?

The formation of arsenic-phytochelatin (As-PC) complexes is thought to be part of the plant detoxification strategy for arsenic. This work examines (i) the arsenic (As) concentration-dependent formation of As-PC complex formation and (ii) redistribution and metabolism of As after arrested As uptake in Helianthus annuus. HPLC with parallel ICP-MS/ES-MS detection was used to identify and quantify the species present in plant extracts exposed to arsenate (As(V)) (between 0 and 66.7 micromol As l-1 for 24 h). At As concentrations below the EC50 value for root growth (22 micromol As l-1) As uptake is exponential, but it is reduced at concentrations above. Translocation between root and shoot seemed to be limited to the uptake phase of arsenic. No redistribution of As between root and shoot was observed after arresting As exposure. The formation of As-PC complexes was concentration-dependent. The amount and number of As-PC complexes increased exponentially with concentration up to 13.7 micromol As l-1. As(III)-PC3 and GS-As(III)-PC2 complexes were the dominant species in all samples. The ratio of PC-bound As to unbound As increased up to 1.3 micromol As l-1 and decreased at higher concentrations. Methylation of inorganic As was only a minor pathway in H. annuus with about 1% As methylated over a 32 d period. The concentration dependence of As-PC complex formation, amount of unbound reduced and oxidized PC2, and the relative uptake rate showed that As starts to influence the cellular metabolism of H. annuus negatively at As concentrations well below the EC50 value determined by more traditional means. Generally, As-PC complexes and PC-synthesis rate seem to be the more sensitive parameters to be studied when As toxicity values are to be estimated.

Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS.

Results: We found that in the heart, t½ of complex I deactivation during ischemia was 10?min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis.

Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting.

Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

Factors influencing acquisition of Burkholderia cepacia complex organisms in patients with cystic fibrosis

Burkholderia cepacia complex organisms are important transmissible pathogens found in cystic fibrosis (CF) patients. In recent years, the rates of cross-infection of epidemic strains have declined due to effective infection control efforts. However, cases of sporadic B. cepacia complex infection continue to occur in some centers. The acquisition pathways and clinical outcomes of sporadic B. cepacia complex infection are unclear. We sought to determine the patient clinical characteristics, outcomes, incidence, and genotypic relatedness for all cases of B. cepacia complex infection at two CF centers. We also sought to study the external conditions that influence the acquisition of infection. From 2001 to 2011, 67 individual organisms were cultured from the respiratory samples of 64 patients. Sixty-five percent of the patients were adults, in whom chronic infections were more common (68%) (P = 0.006). The incidence of B. cepacia complex infection increased by a mean of 12% (95% confidence interval [CI], 3 to 23%) per year. The rates of transplantation and death were similar in the incident cases who developed chronic infection compared to those in patients with chronic Pseudomonas aeruginosa infection. Multilocus sequence typing revealed 50 individual strains from 65 isolates. Overall, 85% of the patients were infected with unique strains, suggesting sporadic acquisition of infection. The yearly incidence of nonepidemic B. cepacia complex infection was positively correlated with the amount of rainfall in the two sites examined: subtropical Brisbane (r = 0.65, P = 0.031) and tropical Townsville (r = 0.82, P = 0.002). This study demonstrates that despite strict cohort segregation, new cases of unrelated B. cepacia complex infection continue to occur. These data also support an environmental origin of infection and suggest that climate conditions may be associated with the acquisition of B. cepacia complex infections.

Disinfection of Burkholderia cepacia complex from non-touch taps in a neonatal nursery

Burkholderia cepacia complex (Bcc) comprises nine closely related species or genomovars. It is an important causative agent of opportunistic infections and waterborne nosocomial infections. B. cepacia (formerly genomovar I) was identified from the blood culture of a baby in our neonatal unit (NU) in March 2005. B. cepacia was isolated four times from clinical specimens since the introduction of non-touch taps in the NU from 2000 to 2005 and only once from 1994 to 2000. Environmental samples were collected from the NU, including tap water from non-touch taps. Clinical and environmental isolates of Bcc were characterized using molecular identification and strain typing. A literature review was undertaken to delineate a method for eradication of Bcc. Several variations for hot water eradication of the organism from the taps were attempted. Genotyping and molecular analysis revealed that tap water isolates were B. cenocepacia which was a different species from the B. cepacia isolated from blood cultures of the neonate. However, B. cenocepacia has been known to cause nosocomial outbreaks and it was eventually eradicated from the NU by using repeated thermal shock (hot water at 65 degrees C for 10 min), changing taps and decolonizing sinks with hypochlorite. Molecular typing is useful in assisting the investigation of Bcc nosocomial infections.

Mitotic Arrest Deficiency Protein 2 (MAD2) and Histone Deacetylase 6 (HDAC6) Present a Complex Relationship in Their Regulation and Expression and Subsequent Impact on Chemoresponsiveness

Introduction:
Ovarian cancer patients presenting with advanced stage (III/IV)
canceraretreatedwithcarboplatinumincombinationwithpaclitaxel.Despitea
significant initial response rate, fewer than 20% of patients become long-term
survivors. We have published that low MAD2 expression levels associate with
reduced progression free survival (PFS) in patients with high-grade serous
epithelial ovarian cancer (EOC). Moreover, we have demonstrated that MAD2
expressionisdown-regulatedbythemicroRNAmiR-433(
Furlong et al., 2011
).
Interestingly, miR-433 also down-regulates HDAC6 (
Simon et al., 2010
), which
uniquely deacetylates
a
-tubulin prior to HDAC6s binding to
b
-tubulin.
In vitro
studies have shown that HDAC6 inhibition in combination with paclitaxel
treatment enhances chemoresistant cancer cell death. To date, an interaction
between MAD2 and HDAC6 has not been reported.
Experimental design:
MAD2 and HDAC6 immunohistochemistry (IHC) and
Western blot analyses were performed to investigate the role of HDAC6 and
MAD2 in chemoresistance to paclitaxel in high-grade serous EOC.
Results and Discussion:
In vitro
experiments demonstrated that overex-
pression of pre-miR-433, which targets MAD2, resulted in down-regulation
of HDAC6 in EOC cell lines. High levels of HDAC6 are co-expressed with
MAD2 in the paclitaxel resistant UPN251 and OVCAR7 cell lines. While, all
4 paclitaxel resistant EOC cell lines express higher levels of miR-433 than
the paclitaxel sensitive A2780 cells, only ovca432 and ovca433 demonstrated
down-regulation of both HDAC6 and MAD2. Paclitaxel binds to
b
-tubulin and
causesmicrotubulepolymerizationinpaclitaxelsensitivecellsasdemonstrated
by tubulin acetylation in A2780 cells. However, paclitaxel failed to cause a
significant acetylation of
a
-tubulin and microtubule stabilisation in the resistant
UPN251 cells. Therefore resistance in this cell line may be mediated by
aberrantly high HDAC6 activity. We have previously shown that MAD2 knock-
down cells are resistant to paclitaxel (
Furlong F., et al., 2011; Prencipe M.,
et al., 2009
). We measured HDAC6 protein expression in MAD2 knockdown
cells and showed that MAD2 knockdown is associated with concomitant
up-regulation of HDAC6. We hypothesise that the up-regulation of HDAC6
by MAD2 knockdown renders cancer cells more resistant to paclitaxel and
increases the invasive potential of these cells. On-going experiments will test
this hypothesis. Lastly we have observed differential MAD2 and HDAC6 IHC
staining intensity in formalin fixed paraffin embedded EOC samples.
In conclusion
, we have reported on a novel interaction between MAD2 and
HDAC6 which may have important consequences for paclitaxel resistant EOC.
Moreover, understanding chemo-responsiveness in ovarian tumours will lead
to improved patient management and treatment options for women diagnosed
with this disease

The complex ion structure of warm dense carbon measured by spectrally resolved x-ray scattering

We present measurements of the complex ion structure of warm dense carbon close to the melting line at pressures around 100 GPa. High-pressure samples were created by laser-driven shock compression of graphite and probed by intense laser-generated x-ray sources with photon energies of 4.75 keV and 4.95 keV. High-efficiency crystal spectrometers allow for spectrally resolving the scattered radiation. Comparing the ratio of elastically and inelastically scattered radiation, we find evidence for a complex bonded liquid that is predicted by ab-initio quantum simulations showing the influence of chemical bonds under these conditions. Using graphite samples of different initial densities we demonstrate the capability of spectrally resolved x-ray scattering to monitor the carbon solid-liquid transition at relatively constant pressure of 150 GPa. Showing first single-pulse scattering spectra from cold graphite of unprecedented quality recorded at the Linac Coherent Light Source, we demonstrate the outstanding possibilities for future high-precision measurements at 4th Generation Light Sources.

14C as a tool to trace terrestrial carbon in a complex lake system: implications for foodweb structure and carbon cycling

Carbon and nitrogen stable isotope analysis (SIA) has identified the terrestrial subsidy of freshwater food-webs but relies on different 13C fractionation in aquatic and terrestrial primary producers. However dissolved inorganic carbon (DIC) is partly comprised of 13C depleted respiration of terrestrial C and ‘old’ C derived from weathering of catchment geology. SIA thus fails to differentiate between the contribution of old and recently fixed terrestrial C. DIC in alkaline lakes is partially derived from weathering of 14C-free carbonaceous bedrock This
yields an artificial age offset leading samples to appear significantly older than their actual age. As such, 14C can be used as a biomarker to identify the proportion of autochthonous C in the food-web. With terrestrial C inputs likely to increase, the origin and utilisation of ‘old’ or ‘recent’ allochthonous C in the food-web can also be determined. Stable isotopes and 14C were measured for biota, particulate organic matter (POM), DIC and dissolved organic carbon (DOC) from Lough Erne, Northern Ireland, a humic but alkaline lake. High winter δ15N values in calanoid zooplankton (δ15N =24‰) relative to phytoplankton and POM (δ15N =6‰ and 12‰ respectively) may reflect several microbial trophic levels between terrestrial C and calanoids. Furthermore winter calanoid 14C ages are consistent with DOC from inflowing rivers (87 and 75 years BP respectively) but not phytoplankton (355 years BP). Summer calanoid δ13N, δ15N and 14C (312 years BP) indicate greater reliance on phytoplankton. There is also temporal and spatial variation in DIC, DOC and POM C isotopes.

14C as a tool to trace terrestrial carbon in a complex lake system: implications for food-web structure and carbon cycling

Globally lakes bury and remineralise significant quantities of terrestrial C, and the associated flux of terrestrial C strongly influences their functioning. Changing deposition chemistry, land use and climate induced impacts on hydrology will affect soil biogeochemistry and terrestrial C export¹ and hence lake ecology with potential feedbacks for regional and global C cycling. C and nitrogen stable isotope analysis (SIA) has identified the terrestrial subsidy of freshwater food webs. The approach relies on different ¹³C fractionation in aquatic and terrestrial primary producers, but also that inorganic C demands of aquatic primary producers are partly met by ¹³C depleted C from respiration of terrestrial C, and ‘old’ C derived from weathering of catchment geology. SIA thus fails to differentiate between the contributions of old and recently fixed terrestrial C. Natural abundance ¹⁴C can be used as an additional biomarker to untangle riverine food webs² where aquatic and terrestrial δ ¹³C overlap, but may also be valuable for examining the age and origin of C in the lake. Primary production in lakes is based on dissolved inorganic C (DIC). DIC in alkaline lakes is partially derived from weathering of carbonaceous bedrock, a proportion of which is¹⁴C-free. The low ¹⁴C activity yields an artificial age offset leading samples to appear hundreds to thousands of years older than their actual age. As such, ¹⁴C can be used to identify the proportion of autochthonous C in the food-web. With terrestrial C inputs likely to increase, the origin and utilisation of ‘fossil’ or ‘recent’ allochthonous C in the food-web can also be determined. Stable isotopes and 14C were measured for biota, particulate organic matter (POM), DIC and dissolved organic carbon (DOC) from Lough Erne, Northern Ireland, a humic alkaline lake. Temporal and spatial variation was evident in DIC, DOC and POM C isotopes with implications for the fluctuation in terrestrial export processes. Ramped pyrolysis of lake surface sediment indicates the burial of two C components. ¹⁴C activity (507 ± 30 BP) of sediment combusted at 400˚C was consistent with algal values and younger than bulk sediment values (1097 ± 30 BP). The sample was subsequently combusted at 850˚C, yielding 14C values (1471 ± 30 BP) older than the bulk sediment age, suggesting that fossil terrestrial carbon is also buried in the sediment. Stable isotopes in the food web indicate that terrestrial organic C is also utilised by lake organisms. High winter δ ¹⁵N values in calanoid zooplankton (δ ¹⁵N = 24%¸) relative to phytoplankton and POM (δ ¹⁵N = 6h and 12h respectively) may reflect several microbial trophic levels between terrestrial C and calanoids. Furthermore winter calanoid 14C ages are consistent with DOC from an inflowing river (75 ± 24 BP), not phytoplankton (367 ± 70 BP). Summer calanoid δ ^13C, δ ¹⁵N and ¹⁴C (345 ± 80 BP) indicate greater reliance on phytoplankton.

¹ Monteith, D.T et al., (2007) Dissolved organic carbon trends resulting from changes in atmospheric deposition chemistry. Nature, 450:537-535

² Caraco, N., et al.,(2010) Millennial-aged organic carbon subsidies to a modern river food web. Ecology,91: 2385-2393.

Fate Hazardous elements in soils surrounding a coal-fired power plant complex

Proceedings of the 13th International UFZ-Deltares Conference on Sustainable Use and Management of Soil, Sediment and Water Resources - 9–12 June 2015 • Copenhagen, Denmark