We determined the mechanisms of calcium signaling in the human ureter, and the relationship to pe... more We determined the mechanisms of calcium signaling in the human ureter, and the relationship to peristaltic contractions and bundular structure in living tissue, thereby advancing the understanding of ureteral function in health and obstruction and reflux. Confocal imaging of 31 ureters was performed and simultaneous force and calcium measurements were made. Immunohistochemistry and Western blotting were also performed. Confocal imaging showed a 3-dimensional network of smooth muscle bundles with no defined longitudinal or circular layers. Fast propagating Ca waves spread throughout the bundles, were closely associated with contraction and depended on L-type Ca channel entry. Immunohistochemistry and Western blotting demonstrated L-type Ca channels, Ca dependent K channels, sarcoplasmic reticulum Ca-adenosine triphosphatase isoforms 2 and 3, inositol triphosphate, and ryanodine receptors. Modulation of Ca and K channel activity was a potent mechanism for affecting Ca and force, whereas manipulation of the sarcoplasmic reticulum had little effect. To our knowledge this study represents the first measurements of Ca signals in the human ureter obtained during phasic contractions and in response to agonists. Results show that it is controlled by fast propagating Ca waves, which spread rapidly between the muscle bundles, producing regular contractions, and drugs that interfere with excitability or Ca entry through L-type Ca channels have profound effects on Ca signaling and contractility. These data are discussed in relation to the treatment of patients with suspected ureteral dysfunction using Ca entry blockers.
The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulat... more The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findings provide novel evidence of a potential link between hypoxia within the hoof and up-regulation of MMPs which may in turn result in damage to the lamellar basement membrane.
We have developed an efficient and inexpensive pipeline for streamlining large-scale collection a... more We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections ...
Introduction. P. aeruginosa is an opportunistic Gram-negative pathogen frequently isolated in uri... more Introduction. P. aeruginosa is an opportunistic Gram-negative pathogen frequently isolated in urinary tract infections (UTI) affecting elderly and catheterized patients and associated with ineffective antibiotic treatment and poor clinical outcomes. Gap statement. Invasion has been shown to play an important role in UTI caused by E. coli but has only recently been studied with P. aeruginosa . The ability of P. aeruginosa to adapt and evolve in chronic lung infections is associated with resistance to antibiotics but has rarely been studied in P. aeruginosa UTI populations. Aim. We sought to determine whether phenotypic and genotypic heterogeneity exists in P. aeruginosa UTI isolates and whether, like urinary pathogenic Escherichia coli , these could invade human bladder epithelial cells – two factors that could complicate antibiotic treatment. Methodology. P. aeruginosa UTI samples were obtained from five elderly patients at the Royal Liverpool University Hospital as part of routine ...
Developmental and tissue-specific differences in isoforms allow Na+, K+-ATPase function to be tig... more Developmental and tissue-specific differences in isoforms allow Na+, K+-ATPase function to be tightly regulated, as they control sensitivity to ions and inhibitors. Uterine contraction relies on the activity of the Na+, K+ATPase, which creates ionic gradients that drive excitation-contraction coupling. It is unknown whether Na+, K+ATPase isoforms are regulated throughout pregnancy or whether they have a direct role in modulating uterine contractility. We hypothesized that gestation-dependent differential expression of isoforms would affect contractile responses to Na+, K+ATPase subunit inhibition with ouabain. Our aims were therefore: (1) to determine the gestation-dependent expression of mRNA transcripts, protein abundance and tissue distribution of Na+, K+ATPase isoforms in myometrium, (2) to investigate the functional effects of differential isoform expression via ouabain sensitivity and, (3) if changes in contractile responses can be explained by changes in intracellular [Ca2+...
Developmental and tissue-specific differences in isoforms allow Na, K-ATPase function to be tight... more Developmental and tissue-specific differences in isoforms allow Na, K-ATPase function to be tightly regulated, as they control sensitivity to ions and inhibitors. Uterine contraction relies on the activity of the Na, KATPase, which creates ionic gradients that drive excitation-contraction coupling. It is unknown whether Na, KATPase isoforms are regulated throughout pregnancy or whether they have a direct role in modulating uterine contractility. We hypothesized that gestation-dependent differential expression of isoforms would affect contractile responses to Na, KATPase subunit inhibition with ouabain. Our aims were therefore: (1) to determine the gestation-dependent expression of mRNA transcripts, protein abundance and tissue distribution of Na, KATPase isoforms in myometrium; (2) to investigate the functional effects of differential isoform expression via ouabain sensitivity; and (3) if changes in contractile responses can be explained by changes in intracellular [Ca]. Changes in ...
We determined the mechanisms of calcium signaling in the human ureter, and the relationship to pe... more We determined the mechanisms of calcium signaling in the human ureter, and the relationship to peristaltic contractions and bundular structure in living tissue, thereby advancing the understanding of ureteral function in health and obstruction and reflux. Confocal imaging of 31 ureters was performed and simultaneous force and calcium measurements were made. Immunohistochemistry and Western blotting were also performed. Confocal imaging showed a 3-dimensional network of smooth muscle bundles with no defined longitudinal or circular layers. Fast propagating Ca waves spread throughout the bundles, were closely associated with contraction and depended on L-type Ca channel entry. Immunohistochemistry and Western blotting demonstrated L-type Ca channels, Ca dependent K channels, sarcoplasmic reticulum Ca-adenosine triphosphatase isoforms 2 and 3, inositol triphosphate, and ryanodine receptors. Modulation of Ca and K channel activity was a potent mechanism for affecting Ca and force, whereas manipulation of the sarcoplasmic reticulum had little effect. To our knowledge this study represents the first measurements of Ca signals in the human ureter obtained during phasic contractions and in response to agonists. Results show that it is controlled by fast propagating Ca waves, which spread rapidly between the muscle bundles, producing regular contractions, and drugs that interfere with excitability or Ca entry through L-type Ca channels have profound effects on Ca signaling and contractility. These data are discussed in relation to the treatment of patients with suspected ureteral dysfunction using Ca entry blockers.
The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulat... more The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findings provide novel evidence of a potential link between hypoxia within the hoof and up-regulation of MMPs which may in turn result in damage to the lamellar basement membrane.
We have developed an efficient and inexpensive pipeline for streamlining large-scale collection a... more We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections ...
Introduction. P. aeruginosa is an opportunistic Gram-negative pathogen frequently isolated in uri... more Introduction. P. aeruginosa is an opportunistic Gram-negative pathogen frequently isolated in urinary tract infections (UTI) affecting elderly and catheterized patients and associated with ineffective antibiotic treatment and poor clinical outcomes. Gap statement. Invasion has been shown to play an important role in UTI caused by E. coli but has only recently been studied with P. aeruginosa . The ability of P. aeruginosa to adapt and evolve in chronic lung infections is associated with resistance to antibiotics but has rarely been studied in P. aeruginosa UTI populations. Aim. We sought to determine whether phenotypic and genotypic heterogeneity exists in P. aeruginosa UTI isolates and whether, like urinary pathogenic Escherichia coli , these could invade human bladder epithelial cells – two factors that could complicate antibiotic treatment. Methodology. P. aeruginosa UTI samples were obtained from five elderly patients at the Royal Liverpool University Hospital as part of routine ...
Developmental and tissue-specific differences in isoforms allow Na+, K+-ATPase function to be tig... more Developmental and tissue-specific differences in isoforms allow Na+, K+-ATPase function to be tightly regulated, as they control sensitivity to ions and inhibitors. Uterine contraction relies on the activity of the Na+, K+ATPase, which creates ionic gradients that drive excitation-contraction coupling. It is unknown whether Na+, K+ATPase isoforms are regulated throughout pregnancy or whether they have a direct role in modulating uterine contractility. We hypothesized that gestation-dependent differential expression of isoforms would affect contractile responses to Na+, K+ATPase subunit inhibition with ouabain. Our aims were therefore: (1) to determine the gestation-dependent expression of mRNA transcripts, protein abundance and tissue distribution of Na+, K+ATPase isoforms in myometrium, (2) to investigate the functional effects of differential isoform expression via ouabain sensitivity and, (3) if changes in contractile responses can be explained by changes in intracellular [Ca2+...
Developmental and tissue-specific differences in isoforms allow Na, K-ATPase function to be tight... more Developmental and tissue-specific differences in isoforms allow Na, K-ATPase function to be tightly regulated, as they control sensitivity to ions and inhibitors. Uterine contraction relies on the activity of the Na, KATPase, which creates ionic gradients that drive excitation-contraction coupling. It is unknown whether Na, KATPase isoforms are regulated throughout pregnancy or whether they have a direct role in modulating uterine contractility. We hypothesized that gestation-dependent differential expression of isoforms would affect contractile responses to Na, KATPase subunit inhibition with ouabain. Our aims were therefore: (1) to determine the gestation-dependent expression of mRNA transcripts, protein abundance and tissue distribution of Na, KATPase isoforms in myometrium; (2) to investigate the functional effects of differential isoform expression via ouabain sensitivity; and (3) if changes in contractile responses can be explained by changes in intracellular [Ca]. Changes in ...
Uploads
Papers by Rachel Floyd