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1.  Electrospun Polyhydroxybutyrate and Poly(L-lactide-co-ε-caprolactone) Composites as Nanofibrous Scaffolds 
BioMed Research International  2014;2014:741408.
Electrospinning can produce nanofibrous scaffolds that mimic the architecture of the extracellular matrix and support cell attachment for tissue engineering applications. In this study, fibrous membranes of polyhydroxybutyrate (PHB) with various loadings of poly(L-lactide-co-ε-caprolactone) (PLCL) were successfully prepared by electrospinning. In comparison to PLCL scaffolds, PLCL blends with PHB exhibited more irregular fibre diameter distributions and higher average fibre diameters but there were no significant differences in pore size. PLCL/PHB scaffolds were more hydrophilic (<120°) with significantly reduced tensile strength (ca. 1 MPa) compared to PLCL scaffolds (150.9 ± 2.8° and 5.8 ± 0.5 MPa). Increasing PLCL loading in PHB/PLCL scaffolds significantly increased the extension at break, (4–6-fold). PLCL/PHB scaffolds supported greater adhesion and proliferation of olfactory ensheathing cells (OECs) than those exhibiting asynchronous growth on culture plates. Mitochondrial activity of cells cultivated on the electrospun blended membranes was enhanced compared to those grown on PLCL and PHB scaffolds (212, 179, and 153%, resp.). Analysis showed that PLCL/PHB nanofibrous membranes promoted cell cycle progression and reduced the onset of necrosis. Thus, electrospun PLCL/PHB composites promoted adhesion and proliferation of OECs when compared to their individual PLCL and PHB components suggesting potential in the repair and engineering of nerve tissue.
PMCID: PMC4034502  PMID: 24900983
2.  Adjuvant epirubicin followed by cyclophosphamide, methotrexate and fluorouracil (CMF) vs CMF in early breast cancer: results with over 7 years median follow-up from the randomised phase III NEAT/BR9601 trials 
British Journal of Cancer  2012;107(8):1257-1267.
The National Epirubicin Adjuvant Trial (NEAT) and BR9601 trials tested the benefit of epirubicin when added to cyclophosphamide, methotrexate and 5-fluorouracil (E-CMF) compared with standard CMF in adjuvant chemotherapy for women with early breast cancer. This report details longer follow-up with interesting additional time-dependent analyses.
National Epirubicin Adjuvant Trial used epirubicin (E) 3-weekly for four cycles followed by classical (c) CMF for four cycles (E-CMF) compared with cCMF for six cycles. BR9601 used E 3-weekly for four cycles followed by CMF 3-weekly for four cycles, compared with CMF 3-weekly for eight cycles.
In all, 2391 eligible patients were randomised and with a median 7.4-year follow-up, E-CMF confirmed a significant benefit over CMF in both relapse-free survival (RFS) (78% vs 71% 5 years RFS, respectively, hazard ratio (HR)=0.75 (95% CI: 0.65–0.86), P<0.0001) and overall survival (OS) (84% vs 78% 5 years OS, respectively, HR=0.76 (95% CI: 0.65–0.89), P=0.0007). Interaction of treatment effect and prognostic factors was demonstrated for duplication of chromosome 17 centromeric enumeration (Ch17CEP) as previously reported. Poor prognostic factors at diagnosis (ER and PR negative and HER2 positive) showed time-dependent annual hazard rates for RFS and OS. In univariate analysis, these factors demonstrated more favourable HRs for RFS after 5 years. Treatment effects also suggested a differential benefit for E-CMF within the first 5 years for poor prognosis tumours.
Longer follow-up has confirmed E-CMF as significantly superior to CMF for all patients. Ch17CEP duplication was the only biomarker that demonstrated significant treatment interaction. Standard poor prognostic factors at diagnosis were time-dependent, and after 5 years disease-free, poor prognosis patients demonstrated favourable HRs for survival.
PMCID: PMC3494422  PMID: 23047592
NEAT; breast cancer; adjuvant chemotherapy; anthracyclines; epirubicin; classical CMF
3.  Coverage and Recovery of Upstream Protein Fractionation Methods in LC-MS/MS Workflows 
The proteome of any cell or even any subcellular fraction remains too complex for complete analysis by one dimension of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hence, to achieve greater depth of coverage for a proteome of interest, most groups routinely subfractionate the sample prior to LC-MS/MS so that the material entering LC-MS/MS is less complex than the original sample. Protein and/or peptide fractionation methods that biochemists have used for decades, such as strong cation exchange chromatography (SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most common prefractionation methods used currently. There has, as yet, been no comprehensive, controlled evaluation of the relative merits of the various methods, although some binary comparisons have been made. We will discuss the most popular methods for fractionating samples at both the protein and peptide level, demonstrating quantitatively which are the best methods for optimal recovery and proteome coverage. A novel approach for fractionating samples at the level of protein complexes will also be discussed.
PMCID: PMC3186557
4.  Survival in Malignant Peripheral Nerve Sheath Tumours: A Comparison between Sporadic and Neurofibromatosis Type 1-Associated Tumours 
Sarcoma  2009;2009:756395.
We studied 123 patients with malignant peripheral nerve sheath tumours (MPNSTs) between 1979 and 2002. However, 90 occurred sporadically whereas 33 were associated with neurofibromatosis type 1 (NF1). Survival was calculated using Kaplan-Meier survival curves and we used Cox's proportional hazards model to identify independent prognostic factors. A 5-year survival for 110 nonmetastatic patients was 54%; (33% NF1 and 63% sporadic P = .015). Tumour stage and site were significant prognostic indicators after univariate analysis. After multivariate analysis, however, only NF1 (P = .007) and tumour volume more than 200 m (P = .015) remained independent predictors of poor outcome. We recommend that NF1 be taken into account during MPNST staging. As the survival rate in the NF group was dependant on tumour volume, routine screening of these patients with FDG PET and/or MRI may be warranted, thereby staging and controlling them at the earliest possible opportunity.
PMCID: PMC2666272  PMID: 19360115
5.  Characterization of the elastic properties of the nuclear envelope 
Underlying the nuclear envelope (NE) of most eukaryotic cells is the nuclear lamina, a meshwork consisting largely of coiled-coil nuclear intermediate filament proteins that play a critical role in nuclear organization and gene expression, and are vital for the structural stability of the NE/nucleus. By confocal microscopy and micromanipulation of the NE in living cells and isolated nuclei, we show that the NE undergoes deformations without large-scale rupture and maintains structural stability when exposed to mechanical stress. In conjunction with image analysis, we have developed theory for a two-dimensional elastic material to quantify NE elastic behaviour. We show that the NE is elastic and exhibits characteristics of a continuous two-dimensional solid, including connections between lamins and the embedded nuclear pore complexes. Correlating models of NE lateral organization to the experimental findings indicates a heterogeneous lateral distribution of NE components on a mesoscopic scale.
PMCID: PMC1578255  PMID: 16849165
mechanics; confocal laser scanning microscopy; micropipette aspiration; green fluorescent protein-lamin A; nucleoporin p62
6.  Alveolar macrophage priming by intravenous administration of chitin particles, polymers of N-acetyl-D-glucosamine, in mice. 
Infection and Immunity  1997;65(5):1734-1741.
Intravenous (i.v.) administration of phagocytosable chitin particles (1 to 10 microm) in C57BL/6 mice and SCID mice primed alveolar macrophages (Mphi) within 3 days to yield up to a 50-fold increase in their oxidative burst when elicited in vitro with phorbol myristate acetate (PMA). C57BL/6 mice pretreated with monoclonal antibodies (MAbs) against mouse gamma interferon (IFN-gamma) or NK1.1 showed a markedly decreased level of alveolar Mphi priming following injection of chitin particles. To confirm IFN-gamma production in vitro, spleen cells isolated from normal C57BL/6 mice and SCID mice were cultured with chitin particles. Significant IFN-gamma production was observed following stimulation with chitin but not with chitosan or latex beads. When spleen cells were treated with anti-NK1.1 MAb, IFN-gamma production was significantly inhibited. Another set of experiments showed that when C57BL/6 mice were pretreated i.v. with a small dose IFN-gamma, a higher level of priming was induced with not only phagocytosable chitin particles but also phagocytosable chitosan and even latex beads. Likewise, the spleen cell cultures preconditioned with IFN-gamma provided an up-regulation of IFN-gamma production by these phagocytosable particles. Taken together, the in vivo and in vitro results suggest that (i) the alveolar Mphi priming mechanism is due, at least in part, to direct activation of Mphi by IFN-gamma, which is produced by NK1.1+ CD4- cells; (ii) IFN-gamma would have an autocrine-like effect on Mphi and make them more responsive to particle priming; and (iii) phagocytosis of particulates, probably by a postmembrane event such as interiorization, appears to be important for the up-regulation of alveolar Mphi priming and IFN-gamma production.
PMCID: PMC175208  PMID: 9125555
8.  Role of CD44 in the reaction of vascular smooth muscle cells to arterial wall injury. 
Journal of Clinical Investigation  1996;97(3):596-603.
CD44, the principal receptor for hyaluronic acid, is a widely distributed cell surface proteoglycan involved in cellular activation, proliferation, and migration. These processes are also central to the vascular smooth muscle cell's response to arterial wall injury. We evaluated the expression of CD44 and its isoform, CD44-V6, on vascular smooth muscle cells in vitro and in vivo and assessed the role of CD44 in DNA synthesis. Cultured vascular smooth muscle cells expressed CD44 and CD44-V6 at levels equal to or higher than those of the beta 1 and beta 2 integrins. In a rat carotid artery balloon injury model, CD44 and CD44-V6 mRNAs were unregulated in vascular smooth muscle cells after injury, and CD44 protein expression was greatest at the luminal edge of the growing neointima. CD44-expressing smooth muscle cells proliferated actively, and hyaluronic acid expression increased after injury in a temporal pattern similar to that of CD44. Through binding to hyaluronic acid, CD44 augmented DNA synthesis in cultured human and rat smooth muscle cells by 48 +/- 7.8 and 100 +/- 12.5%, respectively, an effect inhibited by an anti-CD44 antibody that blocked hyaluronate binding. These observations support a role for CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.
PMCID: PMC507094  PMID: 8609213
9.  bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage. 
Journal of Bacteriology  1995;177(21):6058-6063.
Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.
PMCID: PMC177442  PMID: 7592367
10.  Identification of alcaligin as the siderophore produced by Bordetella pertussis and B. bronchiseptica. 
Journal of Bacteriology  1995;177(4):1116-1118.
The siderophores produced by iron-starved Bordetella pertussis and B. bronchiseptica were purified and were found to be identical. Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans.
PMCID: PMC176712  PMID: 7860593
11.  Survey of state health agencies' staff who practice the epidemiology of noninfectious diseases and conditions. 
Public Health Reports  1994;109(1):112-117.
The primary causes of mortality in the United States are noninfectious diseases and conditions. Epidemiologic and intervention activities related to most of these diseases and conditions have increased in most State health agencies over the past decade. Because little was known of the practice of noninfectious disease epidemiology in State health agencies, a mail survey was undertaken in 1991. Persons working in State health agencies who responded to the survey had a graduate degree in epidemiology, biostatistics, or related fields and actively participated in the epidemiology of noninfectious diseases or conditions. Respondents were from 48 States, predominantly male (56 percent) and white (92 percent). On an average, respondents spent roughly half of their time actually doing epidemiology. The focus of noninfectious disease epidemiology has been categorized by risk factors (environment, occupation, nutrition, tobacco, and substance abuse), diseases (diabetes, cancer, and cardiovascular disease), and health conditions (injury, birth defects, and other reproductive conditions). The percentage of respondents who reported epidemiologic activity in any risk factor, disease, or condition varied from 55 percent for environmental epidemiology to 9 percent in nutritional epidemiology. Respondents from 41 States reported activity in environmental epidemiology, those from 18 States reported activity in substance-abuse epidemiology, and those from 13 States reported activity in nutritional epidemiology. Although the practice of noninfectious disease epidemiology appears to be considered important in the majority of States, the extent of practice varies markedly. Those risk factors, diseases, and conditions that are most frequently associated with morbidity and mortality are the least addressed epidemiologically in State health agencies.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1402250  PMID: 8303004
12.  A siderophore production mutant of Bordetella bronchiseptica cannot use lactoferrin as an iron source. 
Infection and Immunity  1993;61(6):2698-2702.
Bordetella bronchiseptica secreted a hydroxamate siderophore when grown in Fe-depleted medium. A Tn5lac insertion mutant of B. bronchiseptica, DBB22, did not produce this hydroxamate siderophore and was incapable of using lactoferrin as an Fe source. Our data suggest that B. bronchiseptica uses a siderophore for removal of Fe from lactoferrin and transferrin rather than relying upon a receptor for these host Fe-binding proteins.
PMCID: PMC280903  PMID: 8500910
14.  Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement. 
Journal of Bacteriology  1988;170(7):3040-3045.
The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.
PMCID: PMC211246  PMID: 2838456
15.  Uric acid effects on in vitro models of rheumatoid inflammatory and autoimmune processes. 
Annals of the Rheumatic Diseases  1983;42(3):338-342.
A neutrophil monolayer system was used to study the effects of uric acid on neutrophil-aggregate interactions important in rheumatoid inflammation. No effect on immunoglobulin G aggregate phagocytosis was seen, but hyperuricaemic levels of uric acid were associated with an enhancement of phagocytosis-induced release of the azurophilic granular enzyme beta-glucuronidase. A trinitrophenyl-coupled mononuclear leucocyte rheumatoid factor plaque-forming assay was utilised to study uric acid effects on polyclonal activation of immunocompetent cells. Low levels of uric acid enhanced and high levels suppressed this system. Hyperuricaemia may enhance some aspects of rheumatoid inflammation, while uric acid may modulate an important component of rheumatoid autoimmunity.
PMCID: PMC1001143  PMID: 6859966
16.  Clinical tests in aquatic toxicology: state of the art 
Hazard assessment of chemicals to aquatic organisms involves the use of many toxicity tests. Acute toxicity tests, embryo-larval toxicity tests, and chronic toxicity tests that measure survival, growth, and reproductive effects now provide the most relative utility for evaluation of potential chemical hazards to aquatic life. Physiological, biochemical, and histological measurements have a low relative utility as diagnostic tests in aquatic toxicology because it is not yet possible to relate changes in these sublethal responses to adverse environmental impacts. The problem of interpreting the toxicological significance of chemical-induced changes in biochemical and physiological mechanisms is twofold: (1) the understanding of physiological and biochemical regulatory mechanisms in fish is limited; and (2) parallel changes in these mechanisms are difficult to correlate with toxicant exposure and impaired ability of fish to survive. To overcome this problem, more physiological and biochemical research must be conducted in conjunction with toxicity studies that measure important whole-animal responses. Toxicant-induced biochemical and physiological responses must be correlated unequivocally with responses related to reproduction, growth and development, survival, or fish health if pertinent diagnostic tests are to be developed for use in aquatic toxicology. The use of diagnostic tests in hazard assessment procedures can decrease the time required for safety evaluation of chemicals, define no-effect exposure concentrations more adequately, and provide a better understanding of the mode of action of chemicals. Considerations for improving the status of the “state of the art” of diagnostic or clinical tests in aquatic toxicology are discussed.
PMCID: PMC1568523  PMID: 7389682
17.  Anaerobic utilization of phosphite and hypophosphite by Bacillus sp. 
A Bacillus sp. capable of utilizing phosphite and hypophosphite under anaerobic conditions was isolated from Cape Canerval soil samples. The organism was isolated on a glucose-mineral salts medium with phosphate deleted. Anaerobic cultivation of this isolate resulted in decreases in the hypophosphite or phosphite concentration, increases in turbidity, cell count, and dry-cell weight, and decreases in pH and glucose concentration. The optimum hypophosphite concentration for this isolate was 60 microgram/ml, whereas the optimum phosphate concentration was greater than 1,000 microgram/ml, suggesting that higher concentrations of hypophosphite may be toxic to this isolate. Hypophosphite or phosphite utilization was accompanied by little or no detectable accumulation of phosphate in the medium, and 32P-labeled hypophosphite was incorporated into the cell as organic phosphate. When phosphate was present in the medium, the isolate failed to metabolize phosphite. In the presence of phosphite and hypophosphite, the isolate first utilized phosphite and then hypophosphite.
PMCID: PMC242956  PMID: 26310
18.  Response of terrestrial microorganisms to a simulated Martian environment. 
Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 degrees C for 16 h and 24 degrees C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.
PMCID: PMC242914  PMID: 646358
19.  Evaluation of a new microquantity blood collector. 
Journal of Clinical Microbiology  1978;7(3):305-306.
A new device for collecting small amounts of blood from laboratory animals offer distinct advantages over standard syringes during the collection of serum or plasma.
PMCID: PMC274919  PMID: 649764
20.  Dry-heat resistance of selected psychrophiles. 
The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.
PMCID: PMC242613  PMID: 410367
22.  Carcinoma of the Breast 
PMCID: PMC2601330  PMID: 21433677

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