The feasibility of early amniocentesis for fetal karyotyping

This thesis investigates the feasibility of amniocentesis in the first trimester of human pregnancy for the diagnosis of fetal karyotype. A cross sectional study of amniotic fluid from 125 pregnancies at 8–18 weeks' gestation, demonstrated an exponential increase in the total number of cells with gestation; however, the number of viable cells did not change significantly. A subsequent pilot study of amniocentesis in 56 women, prior to elective termination of pregnancy at 9–14 weeks' gestation, demonstrated culture success in all 39 cases where the fetal crown-rump length was >37mm, but in only 8 of the 17 (47%) where it was >37mm. In a randomised study a sample was successfully obtained at the first attempt in 320 of 324 (98.8%) cases of early amniocentesis and in 323 of 326 (99%) cases of chorion villus sampling; culture and chromosomal analysis was successful in 98.1% and 99.4% of these cases respectively. To limit the large proportion of amniotic fluid removed at early amniocentesis amnifiltration was developed. This is the filtration of amniotic fluid to trap free cells whilst the fluid is returned to the amniotic sac. A series of experiments involving 218 patients undergoing elective termination of pregnancy at 10-14 weeks' gestation established that (i), a cellulose acetate filter with 0.8 ?m pore size was the most efficient filter tested (ii) 20 mls amniotic fluid must be filtered to achieve the same number of clones in culture as early amniocentesis, (iii) filtered cells could be cultured and karyotyped in a similar time interval to early amniocentesis, (iv) amnifiltration was not associated with bacterial or viral contamination of the amniotic fluid and did not significantly change the urea, electrolytes, proteins and enzyme concentration . The safety and diagnostic accuracy of early amniocentesis remains to be determined by a randomised trial, but preliminary outcome results demonstrate comparable rates of livebirths following early amniocentesis and chorion villus sampling (92% versus 93.8%). However, fetal losses were higher following early amniocentesis than after chorion villus sampling (5.2% versus 1.2%)

Investigating the effectiveness of simplified labels for safe use communication : the case of household detergents

This study assessed the effectiveness of safety communication on the back labels of hazardous products (with regulatory and safety information as dictated by regulatory requirements), with household detergents as a test case. The potential of simplification to increase label effectiveness was evaluated by comparing the currently used labelling approach with two simplified alternatives. The labels mainly differed in terms of the amount of information and the prominence of pictograms. The generalisability of theoretical insights on the effectiveness of pictograms in safety messages to a more real-life context was tested by (a) realistic labels containing several other information elements besides the safety information and (b) target users who are knowledgeable about the product type. One thousand eight hundred (1,800) respondents participated in an online experiment and were randomly exposed to one of the labels. The positive cognitive and behavioural effects commonly attributed to pictorials could not be confirmed, but positive affective effects did emerge. Specifically, even though participants were asked to carefully read the label, they did not spend enough time to process all the content except for the most simplified label. The results did not show meaningful differences between the three labels in terms of information recall (which was poor for all executions), hazard perceptions and behavioural intentions when confronted with an accident. In contrast to this lack of differentiation in cognitive and behavioural intention effects, we did find a clear difference in the affective measure. A majority of the respondents preferred the simplified safety labels. As such, avoiding information overload, and conveying the information in an easier way by means of more prominent use of pictograms, appeared to be appreciated by consumers of household products, while it did not negatively impact label effectiveness

Breakdown of albumin and haemalbumin by the cysteine protease interpain A, an albuminase of Prevotella intermedia

BACKGROUND: Prevotella intermedia is a Gram-negative black-pigmenting oral anaerobe associated with periodontitis in humans, and has a haem requirement for growth, survival and virulence. It produces an iron porphyrin-containing pigment comprising monomeric iron (III) protoporphyrin IX (Fe(III)PPIX.OH; haematin). The bacterium expresses a 90-kDa cysteine protease termed interpain A (InpA) which both oxidizes and subsequently degrades haemoglobin, releasing haem. However, it is not known whether the enzyme may play a role in degrading other haem-carrying plasma proteins present in the gingival sulcus or periodontal pocket from which to derive haem. This study evaluated the ability of InpA to degrade apo- and haem-complexed albumin. RESULTS: Albumin breakdown was examined over a range of pH and in the presence of reducing agent; conditions which prevail in sub- and supra-gingival plaque. InpA digested haemalbumin more efficiently than apoalbumin, especially under reducing conditions at pH 7.5. Under these conditions InpA was able to substantially degrade the albumin component of whole human plasma. CONCLUSIONS: The data point to InpA as an efficient “albuminase” with the ability to degrade the minor fraction of haem-bound albumin in plasma. InpA may thus contribute significantly to haem acquisition by P. intermedia under conditions of low redox potential and higher pH in the inflamed gingival crevice and diseased periodontal pocket where haem availability is tightly controlled by the host

Economic growth, fossil fuel and non-fossil consumption: a Pooled Mean Group analysis using proxies for capital

This study employs a Pooled Mean Group estimator to examine the nexus between economic growth and fossil and non-fossil fuel consumption for 53 countries between 1990 and 2012. The global sample was divided into four categories: developed exporters, developed importers, developing exporters and developing importers. The purpose of these categories was to observe whether factors unique to these countries influence the relationship between energy consumption and economic growth. With the exception of developing importers, evidence of bi-directional causality between fossil fuel consumption and real GDP across all subsamples is observed. This leads to the conclusion that efforts to directly conserve fossil fuels may harm economic growth. In terms of non-fossil fuel use, the results are more diverse. Bi-directional causality between non-fossil fuel use and real GDP is found in the long and short run for developed importers; bi-directional causality only in the long run for developed exporters; negative long-run causality from real GDP to non-fossil fuels for developing exporters; and long-run causality from non-fossil fuel use to real GDP for developing importers. These results lead to the conclusion that other factors have been responsible for the progress seen in non-fossil fuel use. Thus it is concluded that economic growth on its own is insufficient to promote clean energy development. There is a need for policy makers to create an environment conducive to renewable energy investment

DRP-1 is required for BH3 mimetic-mediated mitochondrial fragmentation and apoptosis

The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs, such as Navitoclax and Venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend on MCL-1 for survival, which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far, while demonstrating early promise in vitro, fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects, we developed a rapid differential scanning fluorimetry (DSF)-based assay, and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested, only A-1210477 induced rapid, concentration-dependent apoptosis, which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-XL, A-1210477 exhibited marked synergy with A-1331852, a BCL-XL specific inhibitor, to induce cell death. Despite this selectivity and potency, A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression, suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However, A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1, and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands, and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis

Pyocyanin, a contributory factor in haem acquisition and virulence enhancement of Porphyromonas gingivalis in the lung

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung

Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides

Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism