Environmental stress in the Gulf of Mexico and its potential impact on public health.

The Deepwater Horizon (DWH) oil spill in the Gulf of Mexico was the largest maritime oil spill in history resulting in the accumulation of genotoxic substances in the air, soil, and water. This has potential far-reaching health impacts on cleanup field workers and on the populations living in the contaminated coastal areas. We have employed portable airborne particulate matter samplers (SKC Biosampler Impinger) and a genetically engineered bacterial reporter system (umu-ChromoTest from EBPI) to determine levels of genotoxicity of air samples collected from highly contaminated areas of coastal Louisiana including Grand Isle, Port Fourchon, and Elmer's Island in the spring, summer and fall of 2011, 2012, 2013 and 2014. Air samples collected from a non-contaminated area, Sea Rim State Park, Texas, served as a control for background airborne genotoxic particles. In comparison to controls, air samples from the contaminated areas demonstrated highly significant increases in genotoxicity with the highest values registered during the month of July in 2011, 2013, and 2014, in all three locations. This seasonal trend was disrupted in 2012, when the highest genotoxicity values were detected in October, which correlated with hurricane Isaac landfall in late August of 2012, about five weeks before a routine collection of fall air samples. Our data demonstrate: (i) high levels of air genotoxicity in the monitored areas over last four years post DWH oil spill; (ii) airborne particulate genotoxicity peaks in summers and correlates with high temperatures and high humidity; and (iii) this seasonal trend was disrupted by the hurricane Isaac landfall, which further supports the concept of a continuous negative impact of the oil spill in this region.

CD44 as a Potential Screening Marker for Preliminary Differentiation Between Congenital Dyserythropoietic Anemia Type II and Hereditary Spherocytosis.

BACKGROUND: Bone marrow examination has been the confirmatory test for congenital dyserythropoietic anemia type II (CDAII). Occasional spherocytes on peripheral blood smear can confound the diagnosis. Since a screening test is still unavailable, we explored the feasibility of using flow cytometry as a preliminary screening method. METHODS: Thirteen monoclonal antibodies with specificities for eight erythrocyte membrane proteins were used in FACS analysis to probe the cellular features of red cells from CDAII, normal adults, hereditary spherocytosis (HS), and cord red cells. Confocal microscopy was performed on normal and CDAII to determine the overall distribution of CD44 and CD47. Their expression levels on cultured erythroblasts were also analyzed. RESULTS: The densely stained band 3 as seen in CDAII in gel electrophoresis was also obtained for Dantu phenotype. Likewise analysis of CDAII cases (n = 26) using the eosin-5'maleimide (EMA) binding test found 57% of patients giving results either positive or in the grey area for HS. Enhanced fluorescence of CD44 was detected in 96% of the CDAII patients, and anti-CD47 binding was also elevated to a lesser degree. Although RNA expressions of CD44 and CD47 in the cultured erythroblasts of normal controls and CDAII were similar, confocal microscopy revealed more CDAII red cells giving elevated fluorescence than normal red cells. CONCLUSIONS: A distinction between CDAII and HS can be made using the EMA Binding test and anti-CD44 binding. Confirmation of CDAII can subsequently be made based on clinical presentation together with either bone marrow examination or DNA sequencing of SEC23B. © 2016 International Clinical Cytometry Society.

The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit.

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.

Molecular and biochemical characterization of xrs mutants defective in Ku80.

The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.

Demographic characteristics of survivors of torture presenting for treatment to a national centre for survivors of torture in Ireland (2001-2012).

OBJECTIVES: The asylum process has received a lot of recent media attention but little has been said about the psychological needs of those seeking or granted asylum. Many asylum seekers have experienced trauma and torture, which is associated with substantial psychiatric and psychological morbidity. The Spiritan Asylum Services Initiative (Spirasi) is Ireland's national treatment centre for survivors of torture. The aim of this study was to examine the demographic profile of those attending Spirasi and to consider potential clinical implications of this. METHODS: We retrospectively analysed demographic data relating to the 2590 individuals who attended Spirasi over a 12-year period (2001-2012 inclusive). RESULTS: The majority of attenders were asylum seekers (88%), male (71%) and from African countries. The mean age was 31.9 years. The rate of new referrals, as a percentage of Ireland's asylum-seeking population, has stabilised at ~6% since 2008. Women are underrepresented among those who attend. CONCLUSIONS: The number of new referrals to Spirasi is lower than expected given international estimates of torture prevalence and the impact this has on mental health. Clinicians working with populations of asylum seekers and refugees should sensitively enquire about such events and be aware of the available services. Female refugees and asylum seekers are underrepresented, especially from Asian and Middle Eastern regions. Psychiatric, psychological and general practice services need to respond flexibly to evolving patterns of migration and address potential barriers to access, especially among female refugees and asylum seekers.

A DNA double-strand break defective fibroblast cell line (180BR) derived from a radiosensitive patient represents a new mutant phenotype.

The 180BR cell line was derived from an acute lymphoblastic leukemia patient who overresponded to radiation therapy and died following radiation morbidity. 180BR cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in 180BR cells. To facilitate a measurement of V(D)J recombination, we have characterized 180BRM, a SV40-transformed line derived from 180BR. 180BRM retains the radiosensitivity and defect in DSB repair characteristic of 180BR. The activities associated with DNA-dependent protein kinase are also normal in 180BRM cells. The ability to carry out V(D)J recombination is comparable in 180BRM and a reference control transformed human cell line, MRC5V1. These results show that 180BR and 180BRM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a defect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that 180BR can arrest at the G1-S and G2-M cell cycle checkpoints after irradiation. These results confirm that 180BR can be distinguished from ataxia telangiectasia.

Combined immunodeficiency associated with increased apoptosis of lymphocytes and radiosensitivity fibroblasts.

Severe immunodeficiency characterized by lymphopenia was found in two siblings, one of whom was examined in detail. The calcium flux, pattern of tyrosine phosphorylation of proteins, and interleukin 2 (IL-2) production and proliferation in response to mitogens suggested that the peripheral blood T cells activated normally. The peripheral blood T cells were shown to have an activated phenotype with increased expression of CD45RO+ and CD95/Fas. Increased spontaneous apoptosis occurred in unstimulated lymphocyte cultures. The elevated apoptosis was not due to alterations in expression or to mutations in Bcl-2, Bcl-X(L), or Flip, nor could the spontaneous apoptosis be prevented by blocking Fas, suggesting that it was independent of Fas signaling. This is the first inherited combined immunodeficiency associated with impaired lymphocyte survival. Fibroblasts derived from the patient showed appreciable radiosensitivity in clonal assays, but apoptosis was not elevated. Our results show that the fibroblasts represent a new radiosensitive phenotype not associated with cell cycle checkpoint defects, V(D)J recombination defects, or elevated chromosome breakage. We suggest that the affected gene plays a role in an undetermined damage response mechanism that results in elevated spontaneous apoptosis in lymphoid cells and radiosensitivity in fibroblasts.

Fanconi anemia C gene product plays a role in the fidelity of blunt DNA end-joining.

Mutations in genes controlling the correct functioning of the replicative, repair and recombination machineries may lead to genomic instability. A high level of spontaneous chromosomal aberrations amplified by treatment with DNA cross-linking agents is the hallmark of Fanconi anemia (FA), an inherited chromosomal instability syndrome associated with cancer proneness. Two of the eight FA genes have been cloned (FAA and FAC), but their function has not yet been defined. The lack of homology with known genes suggests the involvement of FA genes in a novel pathway specific to vertebrates. Using a DNA end-joining assay in cultured cells, we studied the processing of both blunt and cohesive-ended double strand breaks (DSB) in normal and FA cells. The results show that: (i) the overall ligation efficiency is normal in FA lymphoblasts; (ii) in FA-C, error-free processing of blunt-ended DSB is markedly decreased, resulting in a higher deletion frequency and larger deletion size; (iii) the fidelity of processing of blunt-DSB is completely restored in FACC cells (complemented with wild-type FAC gene) and the deletion size shifted to values similar to that observed in normal cells; (iv) the fidelity of cohesive end-joining is not affected in FA cells; (v) activities and/or expression of known factors involved in DSB processing, such as the components of the DNA-PK complex and XRCC4, are normal in FA cells. Our results provide strong evidence that the lack of a functional FAC gene results in loss of fidelity of end-joining, which likely accounts for the FA-C phenotype of chromosome instability. We conclude that FAC, and perhaps all FA gene products, are likely to play a role in the fidelity of end-joining of specific DSB.

Problems in the investigation of an apparent outbreak of coagulase-negative staphylococcal septicaemia following cardiac surgery.

Two hundred and twenty-six hospital staff and patients were investigated for the carriage of gentamicin-resistant coagulase-negative staphylococci (CNS) during an apparent outbreak of infection after cardiac surgery. Of the four index strains from infected wounds, three were indistinguishable. The carriage of similar organisms was widespread, particularly among ITU staff (72%) and patients. Ninety-one of the 296 gentamicin-resistant isolates were further investigated, and of these 33 were indistinguishable from index strains even with the use of specialized techniques. Our experience indicates that in outbreaks of infection caused by gentamicin-resistant CNS, resources should be focused on the interruption of transmission and prevention of introduction of these organisms to susceptible patients.

Indirect method for the determination of aluminium by atomic-absorption spectrometry using an air-acetylene flame.

The enhancement of the atomic-absorption signals of iron, cobalt, nickel and chromium in a fuel-rich air-acetylene flame by small amounts of aluminium makes possible the indirect determination of aluminium in the concentration range 0.01-10 ppm. The optimization of working conditions and the occurrence of interferences are reported.

Effect of cell-seeded hydroxyapatite scaffolds on rabbit radius bone regeneration.

Highly porous hydroxyapatite (HA) scaffolds were developed as bone graft substitutes using a template coating process, characterized, and seeded with bone marrow-derived mesenchymal stem cells (BMSCs). To test the hypothesis that cell-seeded HA scaffolds improve bone regeneration, HA scaffolds without cell seeding (HA-empty), HA scaffolds with 1.5 × 10(4) BMSCs (HA-low), and HA scaffolds with 1.5 × 10(6) BMSCs (HA-high) were implanted in a 10-mm rabbit radius segmental defect model for 4 and 8 weeks. Three different fluorochromes were administered at 2, 4, and 6 weeks after implantation to identify differences in temporal bone growth patterns. It was observed from fluorescence histomorphometry analyses that an increased rate of bone infiltration occurred from 0 to 2 weeks (p < 0.05) of implantation for the HA-high group (2.9 ± 0.5 mm) as compared with HA-empty (1.8 ± 0.8 mm) and HA-low (1.3 ± 0.2 mm) groups. No significant differences in bone formation within the scaffold or callus formation was observed between all groups after 4 weeks, with a significant increase in bone regenerated for all groups from 4 to 8 weeks (28.4% across groups). Although there was no difference in bone formation within scaffolds, callus formation was significantly higher in HA-empty scaffolds (100.9 ± 14.1 mm(3) ) when compared with HA-low (57.8 ± 7.3 mm(3) ; p ≤ 0.003) and HA-high (69.2 ± 10.4 mm(3) ; p ≤ 0.02) after 8 weeks. These data highlight the need for a better understanding of the parameters critical to the success of cell-seeded HA scaffolds for bone regeneration.

Hospital budget and rate regulation: why Colorado failed.

A state legislature created an agency to develop a system for regulating hospital budgets and rates, then terminated it two years later. The agency's chairman analyzes the Colorado experience and what can be learned from it.

Epidural anesthesia for acute pseudo-obstruction of the colon (Ogilvie's syndrome).

With the theory that Ogilvie's syndrome is caused by sympathetic inhibition of contractility in the colon as a basis, 18 patients with acute colonic pseudo-obstruction were enrolled in a treatment program assessing the safety and efficacy of sympathetic blockade, by epidural anesthesia, in those who failed conservative management. In some cases colonoscopy had been performed initially and, if unsuccessful, these patients were referred for treatment with epidural anesthesia. Seven patients recovered with conservative treatment alone. One patient refused further treatment and died of her underlying disease. Five of eight patients treated by epidural anesthesia responded. There were no recurrences following successful epidural anesthesia. Five of eight patients treated by colonoscopic decompression responded. No patients required surgical intervention. One patient suffered a subendocardial infarction during colonoscopy. There were no significant complications from epidural anesthesia. Epidural anesthesia is safe, effective, simple, and well tolerated in the management of Ogilvie's syndrome.

Analysis of large deletions in the HPRT gene of primary human fibroblasts using the polymerase chain reaction.

Spontaneous and X-ray-induced mutants of the HPRT gene were isolated from two primary human fibroblast lines. The limited life-span of the mutants restricted the use of methods requiring large quantities of DNA, and the polymerase chain reaction (PCR) was used in particular to check for the presence of multiple genomic sites in mutant analysis. Robust PCR primers were designed to amplify sites of up to 1 kb, mostly with 1-kb spacings between sites, over the entire 56-kb HPRT gene region. Using PCR, large deletions were found in 43% of independent X-ray-induced mutants, and their breakpoints were localized where these fell within the gene. Anonymous DNA sites in the Xq26 chromosomal region containing HPRT (covering > or = 1.5 Mb) were also amplified by PCR to assess codeletion with HPRT; sites up to 1 Mb distal to the gene (DXS86, DXS10) were codeleted in some mutants, but no mutant was found with loss of a proximal site (DXS79).

Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining.

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs) and in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines. In contrast, mutations in the carboxy terminal region of the protein do not impair DNA end-binding activity but decrease the ability of Ku to activate DNA-PK. To gain insight into important functional domains within DNA-PKcs, we have analysed defective mutants, including the mouse scid cell line, and the rodent mutants, irs-20 and V-3. Mutational changes in the carboxy terminal region have been identified in all cases. Our results strongly suggest that the C-terminus of DNA-PKcs is required for kinase activity.

Medial patellofemoral ligament injury following acute transient dislocation of the patella: MR findings with surgical correlation in 14 patients.

PURPOSE: The purpose of this study was to determine the accuracy of MRI in determining both the extent and the location of injury to the medial patellofemoral ligament (MPFL). METHOD: MR findings were compared to the surgical results of 14 consecutive patients who experienced transient patellar dislocation. Two musculoskeletal radiologists, blinded to the surgical results, retrospectively reviewed the MR studies, and a consensus reading was obtained. RESULTS: Surgery demonstrated complete disruption of the MPFL in 7 of 14 patients (50%), with stretching or partial tearing of the MPFL in the remaining 7 (50%) patients. MRI was 85% sensitive and 70% accurate in detecting MPFL disruption. Vastus medialis obliquus muscle elevation was present in 12 of 14 (85%). CONCLUSION: MRI accurately depicts both the extent and the location of MPFL injury following transient patellar dislocation and can therefore play a significant role in directing surgical management of these patients.

The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype.

Antigens of the Rh blood group system are encoded by 2 homologous genes, RHD and RHCE, that produce 2 red cell membrane proteins. The D-negative phenotype is considered to result, almost invariably, from homozygosity for a complete deletion of RHD. The basis of all PCR tests for predicting fetal D phenotype from DNA obtained from amniocytes or maternal plasma is detection of the presence of RHD. These tests are used in order to ascertain the risk of hemolytic disease of the newborn. We have identified an RHD pseudogene (RHD psi) in Rh D-negative Africans. RHDpsi contains a 37 base pair (bp) insert in exon 4, which may introduce a stop codon at position 210. The insert is a sequence duplication across the boundary of intron 3 and exon 4. RHDpsi contains another stop codon in exon 6. The frequency of RHDpsi in black South Africans is approximately 0.0714. Of 82 D-negative black Africans, 66% had RHDpsi, 15% had the RHD-CE-D hybrid gene associated with the VS+ V- phenotype, and only 18% completely lacked RHD. RHDpsi is present in about 24% of D-negative African Americans and 17% of D-negative South Africans of mixed race. No RHD transcript could be detected in D-negative individuals with RHDpsi, probably as a result of nonsense-mediated mRNA decay. Existing PCR-based methods for predicting D phenotype from DNA are not suitable for testing Africans or any population containing a substantial proportion of people with African ethnicity. Consequently, we have developed a new test that detects the 37 bp insert in exon 4 of RHDpsi. (Blood. 2000; 95:12-18)

Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.