The role of the PSD95/Dlg/ZO-1 (PDZ) binding motif of human papillomavirus type 18 E6 oncoprotein in the virus life cycle

A PSD95/Dlg/ZO-1(PDZ)-binding motif (PBM) in the E6 protein of high-risk, cancer-causing human papillomaviruses (HPV) targets a subset of cellular PDZ domain-containing proteins involved in diverse regulatory processes including cell polarity and proliferation, for proteasome-mediated degradation. Interaction with this select group of PDZ domain-containing proteins is negatively regulated by cAMP-dependent protein kinase (PKA) mediated phosphorylation of the E6 PBM. This thesis has sought to address the hypothesis that the PBM of E6 plays an important role within the HPV life cycle. This study has shown that deletion of the E6 PBM from HPV18 genomes affects the morphology and growth of viral episome-containing human keratinocytes and furthermore links E6 PBM function to viral episome replication (maintenance replication and differentiation-dependent amplification). Loss of negative regulation of the E6 PBM by mutation of the PKA recognition motif was associated with increased cell growth and indeed the growth of wildtype HPV18 genome-containing cells responded to changes in PKA signalling. Constitutive E6 PBM function was also associated with invasion of cells suggesting that malignant progression of HPV-infected cells may be linked to changes in PKA signalling. Modulation of the E6 PBM function in the viral genome-containing cells was associated with a change in protein levels of the PDZ domain-containing protein discs large (hDlg) and changes in the non receptor protein phosphastase PTPN13 specific species

The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers

Stroma-induced Jagged1 expression drives PC3 prostate cancer cell migration:disparate effects of RIP-generated proteolytic fragments on cell behaviour and Notch signaling

The Notch ligand Jagged1 is subject to regulated intramembrane proteolysis (RIP) which yields a soluble ectodomain (sJag) and a soluble Jagged1 intracellular domain (JICD). The full-length Jagged1 protein enhances prostate cancer (PCa) cell proliferation and is highly expressed in metastatic cells. However, little is known regarding the mechanisms by which Jagged1 or its RIP-generated fragments might promote PCa bone metastasis. In the current study we show that bone marrow stroma (BMS) induces Jagged1 expression in bone metastatic prostate cancer PC3 cells and that this enhanced expression is mechanistically linked to the promotion of cell migration. We also show that RIP-generated Jagged1 fragments exert disparate effects on PC3 cell behaviour and Notch signaling. In conclusion, the expression of both the full-length ligand and its RIP-generated fragments must be considered in tandem when attempting to regulate Jagged1 as a possible PCa therapy

Assessing the bioactive profile of anti-fungal loaded calcium sulfate against fungal biofilms

Calcium sulfate (CS) has been used clinically as a bone or void filling biomaterial, and due to its resorptive properties have provided the prospect for its use as a release mechanism for local antibiotics to control biofilms. Here, we aimed to test CS beads loaded with three antifungal drugs against planktonic and sessile fungal species to assess whether these antifungal beads could be harnessed to provide consistent release of antifungals at biofilm inhibitive doses. A panel of different fungal species (n=15) were selected for planktonic broth microdilution testing with fluconazole (FLZ), amphotericin B (AMB) and caspofungin (CSP). After establishing planktonic inhibition, antifungal CS beads were introduced to fungal biofilms (n=5) to assess biofilm formation and cell viability through a combination of standard quantitative and qualitative biofilm assays. Inoculation of a hydrogel substrate, packed with antifungal CS beads, was also used to assess diffusion through a semi-dry material, to mimic active infection in-vivo. In general, antifungals released from CS loaded beads were all effective at inhibiting the pathogenic fungi over 7-days within standard MIC ranges for these fungi. We observed a significant reduction of pre-grown fungal biofilms across key fungal pathogens following treatment, with visually observable changes in cell morphology and biofilm coverage provided by scanning electron microscopy. Assessment of biofilm inhibition also revealed reductions in total and viable cells across all organisms tested. These data show that antifungal loaded CS beads produce a sustained antimicrobial effect, which inhibits and kills clinically relevant fungal species in-vitro as planktonic and biofilm cells

The E1 copper binding domain of full-length amyloid precursor protein promotes epithelial to mesenchymal transition in DU145 cells in isoform-specific manner

Epithelial to mesenchymal transition (EMT) confers migratory and dynamic properties on cells and, as such, plays a pivotal role in the development of metastatic, castration resistant prostate cancer. The amyloid precursor protein (APP), although most closely associated with the neurodegenerative condition Alzheimer's disease, has also been linked to the pathogenesis and prognosis of several cancers including prostate cancer. Aims: To investigate whether over-expression of APP could promote EMT in prostate cancer (PCa) DU145 cells and to determine the molecular prerequisites for this effect. Methodology: A range of APP molecular constructs were stably expressed in DU145 cells and their effects on EMT were monitored by morphological analysis and by immunoblotting for the EMT marker proteins, E-cadherin and vimentin. Results: Our results show that the full-length 695 amino acid isoform (APP695), but not APP751 or APP770, promoted EMT via a mechanism requiring an intact extracellular E1 copper binding domain and tyrosine687 within the cytosolic domain of the protein. Conclusion: Targeting the expression of APP695 or the E1 copper binding domain of the protein may, therefore, contribute to therapeutic strategies for the delay or prevention of prostate cancer metastasis

Differential regulation of E-cadherin expression by the soluble ectodomain and intracellular domain of jagged1

Aberrant Jagged1-mediated Notch activation is linked to cancer and induces epithelial-to-mesenchymal transition through the repression of E-cadherin transcription. All three proteins are subject to sequential proteolytic events referred to as regulated intramembrane proteolysis. This process releases soluble protein ectodomains from the cell and, concomitantly, generates intracellular domains capable of nuclear translocation and transcriptional regulation. Aim: To determine the cognate roles of the Jagged1 ectodomain and intracellular domain fragments in the regulation of E-cadherin expression. Methodology: Human embryonic kidney cells were stably transfected with coding DNA constructs analogous to full-length Jagged1, the soluble Jagged1 ectodomain, or the intracellular domain fragment of the protein. Correct construct expression and processing were confirmed by immunoblot analysis of transfectant cell lysates and conditioned culture medium. The effects of the various Jagged1 constructs on endogenous E-cadherin expression and processing were subsequently monitored by immunoblot and RT-qPCR analyses. Results: Both full-length Jagged1 and the soluble Jagged1 ectodomain construct down-regulated E-cadherin expression at the protein and RNA level. In contrast, the Jagged1 intracellular domain fragment construct enhanced E-cadherin expression but only at the RNA level. Conclusion: The soluble Jagged1 ectodomain is sufficient for the down-regulation of E-cadherin expression whereas the intracellular domain of the protein does not exhibit such an effect and actually increases E-cadherin RNA expression. These results raise the interesting possibility of E-cadherin regulation in cells distal to the site of soluble Jagged1 ligand generation

In-vitro efficacy of antibiotics released from calcium sulfate bone void filler beads

Fifteen different antibiotics were individually mixed with a commercially available calcium sulfate bone void filler beads. The antibiotics were: amikacin, ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, colistamethate sodium, daptomycin, gentamicin, imipenem/cilastatin, meropenem, nafcillin, rifampicin, teicoplanin, tobramycin and vancomycin. The efficacy of specific released antibiotics were validated by zone of inhibition (ZOI) testing using a modified Kirby—Bauer disk diffusion method against common periprosthetic joint infection pathogens. With a subset of experiments (daptomycin, rifampin, vancomycin alone and rifampin and vancomycin in combination) we investigated how release varied over 15 days using a repeated ZOI assay. We also tested the ability of these beads to kill biofilms formed by Staphylococcus epidermidis 35984, a prolific biofilm former. The results suggest that certain antibiotics can be combined and released from calcium sulfate with retained antibacterial efficacy. The daptomycin and rifampin plus vancomycin beads showed antimicrobial efficacy for the full 15 days of testing and vancomycin in combination with rifampin prevented resistant mutants. In the biofilm killing assay all of the antibiotic combinations showed a significant reduction in biofilm bacteria after 24 hr. The exposure time was an important factor in the amount of killing and varied among the antibiotics

Antifungal loaded calcium sulfate beads as a potential therapeutic in combating Candida auris

Candida auris provides a substantial global nosocomial threat clinically. With the recent emergence that the organism can readily colonize skin niches, it will likely continue to pose a risk in healthcare units, particularly to patients undergoing surgery. The purpose of this study was to investigate the efficacy of antifungal loaded calcium sulfate beads in combatting C. auris infection. We demonstrate that the CS-packed beads have the potential to interfere with planktonic and sessile C. auris

Killing of a Multispecies Biofilm Using Gram-Negative and Gram-Positive Targeted Antibiotic Released from High Purity Calcium Sulfate Beads

Background: Multispecies biofilm orthopedic infections are more challenging to treat than mono-species infections. In this in-vitro study, we aimed to determine if a multispecies biofilm, consisting of Gram positive and negative species with different antibiotic susceptibilities could be treated more effectively using high purity antibiotic-loaded calcium sulfate beads (HP-ALCSB) containing vancomycin (VAN) and tobramycin (TOB) in combination than alone. Methods: Three sets of species pairs from bioluminescent strains of Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) and clinical isolates, Enterococcus faecalis (EF) and Enterobacter cloacae were screened for compatibility. PA + EF developed intermixed biofilms with similar cell concentrations and so were grown on 316L stainless steel coupons for 72 h or as 24 h agar lawn biofilms and then treated with HP-ALCSBs with single or combination antibiotics and assessed by viable count or bioluminescence and light imaging to distinguish each species. Replica plating was used to assess viability. Results: The VAN + TOB bead significantly reduced the PA + EF biofilm CFU and reduced the concentration of surviving antibiotic tolerant variants by 50% compared to single antibiotics. Conclusions: The combination of Gram-negative and positive targeted antibiotics released from HP-ALCSBs may be more effective in treating multispecies biofilms than monotherapy alone