Elucidation of the K32 Capsular Polysaccharide Structure and Characterization of the KL32 Gene Cluster of Acinetobacter baumannii LUH5549.

Capsular polysaccharide (CPS), isolated from Acinetobacter baumannii LUH5549 carrying the KL32 capsule biosynthesis gene cluster, was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K32 CPS was found to be composed of branched pentasaccharide repeats (K units) containing two residues of ß-D-GalpNAc and one residue of ß-D-GlcpA (ß-D-glucuronic acid) in the main chain and one residue each of ß-D-Glcp and α-D-GlcpNAc in the disaccharide side chain. Consistent with the established CPS structure, the KL32 gene cluster includes genes for a UDP-glucose 6-dehydrogenase (Ugd3) responsible for D-GlcA synthesis and four glycosyltransferases that were assigned to specific linkages. Genes encoding an acetyltransferase and an unknown protein product were not involved in CPS biosynthesis. Whilst the KL32 gene cluster has previously been found in the global clone 2 (GC2) lineage, LUH5549 belongs to the sequence type ST354, thus demonstrating horizontal gene transfer between these lineages.

Improving the finite element model accuracy of tissue engineering scaffolds produced by selective laser sintering.

In bone tissue engineering, both geometrical and mechanical properties of a scaffold play a major part in the success of the treatment. The mechanical stresses and strains that act on cells on a scaffold in a physiological environment are a determining factor on the subsequent tissue formation. Computational models are often used to simulate the effect of changes of internal architectures and external loads applied to the scaffold in order to optimise the scaffold geometry for the prospective implantation site. Finite element analysis (FEA) based on computer models of the scaffold is a common technique, but would not take into account actual inaccuracies due to the manufacturing process. Image based FEA using CT scans of fabricated scaffolds can provide a more accurate analysis of the scaffold, and was used in this work in order to accurately simulate and predict the mechanical performance of bone tissue engineering scaffolds, fabricated using selective laser sintering (SLS), with a view to generating a methodology that could be used to optimise scaffold design. The present work revealed that an approach that assumes isotropic properties of SLS fabricated scaffolds will lead to inaccurate predictions of the FE model. However, a dependency of the grey value of the CT scans and the mechanical properties was discovered, which may ultimately lead to accurate FE models without the need of experimental validation.

Reconstructing the fall: individual, behavioural and contextual factors associated with falls in individuals with intellectual disability.

BACKGROUND: Falls are a significant and recurrent problem for individuals with intellectual disability (ID). There has been little exploration of the fall event from the perspective of the individual who falls or their carers. Research has focused predominantly on personal risk factors, leaving the behavioural and contextual analysis of falls much less understood. This study aimed to identify these additional factors as well as briefly explore the fall experience for individuals and their carers. METHOD: A qualitative design was used incorporating fall reconstructions and ethnographic-style interviews conducted in the home setting. Nine people with ID and their carers/family member participated: five pairs were living at home and four were in out-of-family-home settings. Interviews were recorded, transcribed verbatim and major themes identified via thematic analysis. RESULTS: We identified 17 themes that contributed to falls and fell under the three headings of individual, behavioural or contextual factors. Themes include decreased physical capacity, unsafe behaviours, limited hazard awareness and the impact of others in the home on an individual's fall behaviours. Additionally, families and individuals identified a number of consequences and adaptations which they implemented to alleviate possible fall impact. CONCLUSIONS: Qualitative interviews, observational methods and carer assistance are valuable in offering insight into understanding the individual, behavioural and contextual factors associated with falls in people with ID. The fall reconstruction technique may be a useful supplement when evaluating intrinsic risk in programmes designed to reduce falls.

Myocardial injury: quantitation by cell sorting initiated with antimyosin fluorescent spheres.

Spheres coated with antibodies specific for myosin were used to detect myocardial cell membrane disruption by scanning electron microscopy. Injury in a population of cultured myocytes as then followed and measured by fluorescence-activated cell sorting. This approach provides a unique method for quantitating the evolution of myocardial injury and potentially for assessing the efficacy of interventions aimed at myocardial protection.

A general method for site-specific incorporation of unnatural amino acids into proteins.

A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme beta-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.

Molecular characterization of helix-loop-helix peptides.

A class of regulators of eukaryotic gene expression contains a conserved amino acid sequence responsible for protein oligomerization and binding to DNA. This structure consists of an arginine- and lysine-rich basic region followed by a helix-loop-helix motif, which together mediate specific binding to DNA. Peptides were prepared that span this motif in the MyoD protein; in solution, they formed alpha-helical dimers and tetramers. They bound to DNA as dimers and their alpha-helical content increased on binding. Parallel and antiparallel four-helix models of the DNA-bound dimer were constructed. Peptides containing disulfide bonds were engineered to test the correctness of the two models. A disulfide that is compatible with the parallel model promotes specific interaction with DNA, whereas a disulfide compatible with the antiparallel model abolishes specific binding. Electron paramagnetic resonance (EPR) measurements of nitroxide-labeled peptides provided intersubunit distance measurements that also supported the parallel model.

Finite element predictions compared to experimental results for the effective modulus of bone tissue engineering scaffolds fabricated by selective laser sintering.

A current challenge in bone tissue engineering is to create scaffolds with suitable mechanical properties, high porosity, full interconnectivity and suitable pore size. In this paper, polyamide and polycaprolactone scaffolds were fabricated using a solid free form technique known as selective laser sintering. These scaffolds had fully interconnected pores, minimized strut thickness, and a porosity of approximately 55%. Tensile and compression tests as well as finite element analysis were carried out on these scaffolds. It was found that the values predicted for the effective modulus by the FE model were much higher than the actual values obtained from experimental results. One possible explanation for this discrepancy, viz. the surface roughness of the scaffold and the presence of micropores in the scaffold struts, was investigated with a view to making recommendations on improving FE model configurations for accurate effective property predictions.

An amelanotic malignant melanoma masquerading as hypergranulation tissue.

We report a case of amelanotic malignant melanoma occurring in the nail sulcus of the hallux, which on initial presentation was mistaken for hypergranulation tissue due to an in-growing toenail. We highlight the importance of this differential diagnosis as such lesions can have serious sequelae.

Site-specific mutagenesis with unnatural amino acids.

The incorporation of unnatural amino acids into proteins by site-specific mutagenesis provides a valuable new methodology for the generation of novel proteins that possess unique structural and functional features.

Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy.

The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan(R) reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.

The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration.

The backbone dynamics of the pleckstrin homology (PH) domain from dynamin were studied by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N [1H] nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 microM, and by molecular dynamics (MD) simulations. The analysis was performed using the model-free approach. The method was extended in order to account for observed partial (equilibrium) dimerization of the protein at NMR concentrations. A model is developed that takes into account both rapid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. Internal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S2 approximately 0.9) and from the local correlation time < 100 ps. Of the four extended loop regions that are disordered in the NMR-derived solution structure of the protein, loops beta 1/beta 2 and beta 5/beta 6 are involved in a large-amplitude (S2 down to 0.2 to 0.3) subnanosecond to nanosecond time-scale motion. Reorientation of the loops beta 3/beta 4 and beta 6/beta 7, in contrast, is restricted, characterized by the values of order parameter S2 approximately 0.9 more typical of the protein core. These loops, however, are involved in much slower processes of motion resulting in a conformational exchange on a microsecond to submillisecond time scale. The motions of the terminal regions (residues 1 to 10, 122 to 125) are practically unrestricted (S2 down to 0.05, characteristic times in nanosecond time scale), suggesting that these parts of the sequence do not participate in the protein fold. The analysis shows a larger sensitivity of the 15N relaxation data to protein microdynamic parameters (S2, tau loc) when protein molecular mass (tau c) increases. The use of negative values of the steady state 15N[1H] NOEs as an indicator of the residues not belonging to the folded structure is suggested. The amplitudes of local motion observed in the MD simulation are in a good-agreement with the NMR data for the amide NH groups located in the protein core.

Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity.

It has recently been suggested that pleckstrin homology (PH) domains bind specifically to phospholipids, with phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) being most strongly bound. This observation suggests that PH domains may be responsible for membrane association of proteins in which they occur. Further, this membrane association may be regulated by enzymes that modify lipid head groups to which PH domains may bind. We have studied the binding of phospholipids to the PH domain of human dynamin, a 100 kDa GTPase that is involved in the initial stages of endocytosis. We describe a rapid method for screening PH domain/ligand interactions that gives precise binding constants. We confirm that PtdIns(4,5)P2 can bind to dynamin PH domain, although not in an aggregated state. Using NMR spectroscopy, we have mapped a specific site on the surface of dynamin PH domain of which binding of gIns(1,4,5)P3 (the head-group skeleton of PtdIns(4,5)P2) occurs. The relative affinity of acidic phospholipids for dynamin PH domain correlates with their ability to activate the GTPase of dynamin. We propose, therefore, that the interaction of these phospholipids with dynamin is likely to occur via the PH domain. Given the fact that PH domains are often found in proteins associated with GTPase activity, or in guanine nucleotide exchange factors, we suggest that one role of PH domains may be to couple phosphatidylinositol signalling to GTP hydrolysis.

Solution structure and dynamics of the bioactive retroviral M domain from Rous sarcoma virus.

A biologically active construct of the retroviral M domain from the avian Rous sarcoma virus is defined and its solution structure described. This M domain is fully active in budding and infectivity without myristylation. In spite of a sequence homology level that suggests no relationship among M domains and the family of matrix proteins in mammalian retroviruses, the conserved structural elements of a central core allow an M domain sequence motif to be described for all retroviruses. The surface of the M domain has a highly clustered positive patch comprised of sequentially distant residues. An analysis of the backbone dynamics, incorporating rotational anisotropy, is used to estimate the thermodynamics of proposed domain oligomerization.

ret/PTC and BRAF act as distinct molecular, time-dependant triggers in a sporadic Irish cohort of papillary thyroid carcinoma.

The purpose of this study was to assess BRAF mutation rates in various thyroid tissues and to investigate if concomitant mutations with ret/PTC activation occurred in inflammatory and neoplastic lesions. To this end, we developed a novel Taqman based screening assay for the common T1799A BRAF mutation. Heterozygous T1799A mutations were detected in 13 of 34 (44%) papillary thyroid carcinomas (PTCs) tested. No such mutations were detected in the other tissue types tested. Concomitant presence of both oncogenes was reported in 5 of the 34 PTCs. A significant temporal trend was observed, with ret/PTC chimera detected for the most part before 1997 and BRAF mutations being more prevalent after 1997. The results suggest that some environmental/etiological agent(s) may have influenced the pathobiology of thyroid tumor development, among the population examined, over time.

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.

Role of bipedal lymphangiogram in radiation treatment planning for cervix cancer.

PURPOSE: The role of bipedal lymphangiogram in tailoring radiation portals in radical radiation therapy for Stages II or III cervix cancer is investigated. METHODS AND MATERIALS: The records and simulation films of 87 patients with Stage II or III carcinoma of the cervix treated with radical radiation therapy alone have been retrospectively reviewed. RESULTS: Sixty-two percent of patients who had a bipedal lymphangiogram, subsequently had their radiation fields altered from a "standard portal." The most frequently altered fields were the lateral margin of the postero anterior field and the anterior margin of the lateral fields. In order to cover the lymphatic channels in the pelvis in 90% of cases as outlined by the bipedal lymphangiogram, the lateral margins of the postero anterior fields would need to be 2.5 cm lateral to the pelvic brim and the anterior border of the lateral field, 0.5 cm anterior to the pubic symphysis. CONCLUSION: "Standard" fields adequately cover the median distribution of lymphatics, but may result in a geographic miss in some patients. Bipedal lymphangiograms allow a more accurate tailoring of pelvic lymphatic fields.

Sequential imaging of indium-111-labeled monoclonal antibody in human mammary tumors hosted in nude mice.

Using a bifunctional chelating agent, indium-111 was attached to a monoclonal antibody 10- 3D2 , specific for a 126-kilodalton phosphoglycoprotein antigen associated with human mammary carcinoma, and was then used to localize and visualize human mammary tumors hosted in nude mice. Simultaneous tumor concentration of In-111-10- 3D2 was eight times greater than that of control I-125-MOPC-21. Uptake of F(ab')2 and Fab of 10- 3D2 was also compared. the scintigrams demonstrated that intact antibody provided the best images. Control In-111-labeled MOPC-21 and plasma did not show specific localization in the tumor. Uptake of In-111-labeled 10- 3D2 was also compared in two lines of human mammary tumors, BT-20 and HS- 578T . Imaging with 10- 3D2 was better for BT-20 than for HS- 578T . These studies demonstrated that (a) In-111-10- 3D2 can be utilized to image human mammary tumors hosted in nude mice; (b) intact antibody provided the best tumor images, although F(ab')2 had optimal target-to-background ratios for earlier imaging; and (c) different mammary tumor lines with possibly different concentrations of tumor-associated antigen showed different rates of uptake and apparent saturation with 10- 3D2 .

Apparent symptom overreporting in combat veterans evaluated for PTSD.

Psychometric studies have consistently shown that combat veterans evaluated for posttraumatic stress disorder (PTSD) appear to overreport psychopathology as exhibited by (a) extreme and diffuse levels of psychopathology across instruments measuring different domains of mental illness, and (b) extreme elevations on the validity scale of the MMPI-MMPI-2, in a "fake-bad" direction. The phenomenon of this ubiquitous presentational style is not well understood at present. In this review we describe and delineate the assessment problem posed by this apparent symptom overreporting, and we review the literature regarding several potential explanatory factors. Finally, we address conceptual and practical issues relevant to reaching a better understanding of the phenomenon, and ultimately the clinical syndrome of combat-related PTSD, in both research and clinical settings.

Does EMDR work? And if so, why?: a critical review of controlled outcome and dismantling research.

Research on Eye Movement Desensitization and Reprocessing therapy (EMDR) was reviewed to answer the questions "Does EMDR work?" and "If so, Why?" This first question was further subdivided on the basis of the control group: (a) no-treatment (or wait list control), (b) nonvalidated treatments, and (c) other validated treatments. The evidence supports the following general conclusions: First, EMDR appears to be effective in reducing at least some indices of distress relative to no-treatment in a number of anxiety conditions, including posttraumatic stress disorder, panic disorder, and public-speaking anxiety. Second, EMDR appears at least as effective or more effective than several nonvalidated treatments (e.g., relaxation, active listening) for posttraumatic stress reactions. Third, despite statements implying the contrary, no previously published study has directly compared EMDR with an independently validated treatment for posttraumatic stress disorder (e.g., therapist-directed flooding). In the treatment of simple phobia, participant modeling has been found to be more effective than EMDR. Fourth, our review of dismantling studies reveals there is no convincing evidence that eye movements significantly contribute to treatment outcome. Recommendations regarding further research directions are provided.

Catheter infection control in parenteral nutrition.

Catheter sepsis rates related to total parenteral nutrition are variable and depend on several patient-specific factors. These factors include the presence of immunosuppression or critical illness, the use of multiple intravascular catheters, and bacterial translocation. Catheter-related sepsis may present in the patient as fever, chills, change in mental status, hypotension, and leukocytosis. In patients with suspected catheter-related infection whose peripheral blood cultures do not grow the same organism as a blood culture drawn from the catheter, a guidewire exchange of the catheter has been shown to be effective. This technique should be considered a surgical procedure. Complications that are associated with guidewire exchange of central venous catheters are catheter malposition, embolism of air or septic thrombi, and cardiac arrhythmias.