USP17 is required for peripheral trafficking of lysosomes.

Expression of the deubiquitinase USP17 is induced by multiple stimuli, including cytokines (IL-4/6), chemokines (IL-8, SDF1), and growth factors (EGF), and several studies indicate it is required for cell proliferation and migration. However, the mechanisms via which USP17 impacts upon these cellular functions are unclear. Here, we demonstrate that USP17 depletion prevents peripheral lysosome positioning, as well as trafficking of lysosomes to the cell periphery in response to EGF stimulation. Overexpression of USP17 also increases secretion of the lysosomal protease cathepsin D. In addition, USP17 depletion impairs plasma membrane repair in cells treated with the pore-forming toxin streptolysin O, further indicating that USP17 is required for lysosome trafficking to the plasma membrane. Finally, we demonstrate that USP17 can deubiquitinate p62, and we propose that USP17 can facilitate peripheral lysosome trafficking by opposing the E3 ligase RNF26 to untether lysosomes from the ER and facilitate lysosome peripheral trafficking, lysosome protease secretion, and plasma membrane repair.

A frog-derived antimicrobial peptide as a potential anti-biofilm agent in combating Staphylococcus aureus skin infection.

Staphylococcus aureus (S. aureus), one of the most prevalent bacteria found in atopic dermatitis lesions, can induce ongoing infections and inflammation by downregulating the expression of host defence peptides in the skin. In addition, the emergence of the 'superbug' Methicillin-resistant S. aureus (MRSA) has made the treatment of these infections more challenging. Antimicrobial peptides (AMPs), due to their potent antimicrobial activity, limited evidence of resistance development, and potential immunomodulatory effects, have gained increasing attention as potential therapeutic agents for atopic dermatitis. In this study, we report a novel AMP, brevinin-1E-OG9, isolated from the skin secretions of Odorrana grahami, which shows potent antibacterial activity, especially against S. aureus. Based on the characteristics of the 'Rana Box', we designed a set of brevinin-1E-OG9 analogues to explore its structure-activity relationship. Brevinin-1E-OG9c-De-NH2 exhibited the most potent antimicrobial efficacy in both in vitro and ex vivo studies and attenuated inflammatory responses induced by lipoteichoic acid and heat-killed microbes. As a result, brevinin-1E-OG9c-De-NH2 might represent a promising candidate for the treatment of S. aureus skin infections.

Exploiting the anticancer effects of a nitrogen bisphosphonate nanomedicine for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an incurable aggressive brain cancer in which current treatment strategies have demonstrated limited survival benefit. In recent years, nitrogen-containing bisphosphonates (N-BPs) have demonstrated direct anticancer effects in a number of tumour types including GBM. In this study, a nano-formulation with the RALA peptide was used to complex the N-BP, alendronate (ALN) into nanoparticles (NPs) < 200 nm for optimal endocytic uptake. Fluorescently labelled AlexaFluor®647 Risedronate was used as a fluorescent analogue to visualise the intracellular delivery of N-BPs in both LN229 and T98G GBM cells. RALA NPs were effectively taken up by GBM where a dose-dependent response was evidenced with potentiation factors of 14.96 and 13.4 relative to ALN alone after 72 h in LN229 and T98G cells, respectively. Furthermore, RALA/ALN NPs at the IC50, significantly decreased colony formation, induced apoptosis and slowed spheroid growth in vitro. In addition, H-Ras membrane localisation was significantly reduced in the RALA/ALN groups compared to ALN or controls, indicative of prenylation inhibition. The RALA/ALN NPs were lyophilised to enhance stability without compromising the physiochemical properties necessary for functionality, highlighting the suitability of the NPs for scale-up and in vivo application. Collectively, these data show the significant potential of RALA/ALN NPs as novel therapeutics in the treatment of GBM.

Cathepsin V suppresses GATA3 protein expression in luminal A breast cancer.

BACKGROUND: Lysosomal cysteine protease cathepsin V has previously been shown to exhibit elevated expression in breast cancer tissue and be associated with distant metastasis. Research has also identified that cathepsin V expression is elevated in tumour tissues from numerous other malignancies, but despite this, there has been limited examination of the function of this protease in cancer. Here we investigate the role of cathepsin V in breast cancer in order to delineate the molecular mechanisms by which this protease contributes to tumourigenesis. METHODS: Lentiviral transductions were used to generate shRNA cell line models, with cell line validation undertaken using RQ-PCR and Western blotting. Phenotypic changes of tumour cell biology were examined using clonogenic and invasion assays. The relationship between GATA3 expression and cathepsin V was primarily analysed using Western blotting. Site-directed mutagenesis was used to generate catalytic mutant and shRNA-resistant constructs to confirm the role of cathepsin V in regulating GATA3 expression. RESULTS: We have identified that elevated cathepsin V expression is associated with reduced survival in ER-positive breast cancers. Cathepsin V regulates the expression of GATA3 in ER-positive breast cancers, through promoting its degradation via the proteasome. We have determined that depletion of cathepsin V results in elevated pAkt-1 and reduced GSK-3ß expression, which rescues GATA3 from proteasomal degradation. CONCLUSIONS: In this study, we have identified that cysteine protease cathepsin V can suppress GATA3 expression in ER-positive breast cancers by facilitating its turnover via the proteasome. Therefore, targeting cathepsin V may represent a potential therapeutic strategy in ER-positive breast cancers, by restoring GATA3 protein expression, which is associated with a more favourable clinical outcome.

Controlled coupling of an ultrapotent auristatin warhead to cetuximab yields a next-generation antibody-drug conjugate for EGFR-targeted therapy of KRAS mutant pancreatic cancer.

BACKGROUND: Antibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC. METHODS: In vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens. RESULTS: Site-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells. CONCLUSIONS: Together, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

Identification and target-modifications of temporin-PE: A novel antimicrobial peptide in the defensive skin secretions of the edible frog, Pelophylax kl. esculentus.

A potent natural antimicrobial peptide named temporin-PE was identified and encoded from the skin secretions of Pelophylax kl. esculentus via "shotgun" cloning and LC-MS/MS fragmentation analysis. Target-modifications were carried out to further enhance the antimicrobial and anti-proliferative bioactivities, whilst decreasing the hemolytic effect. A range of bioassays demonstrated that replacing a proline with a tyrosine residue resulted in a loss of the bioactivity against Gram-negative bacteria, but dramatically improved the hemolytic and anti-proliferative activity, indicating the FLP- motif influences the hemolytic activity of temporins. Moreover, the coupling of TAT to the peptide dramatically improved its antimicrobial activity, indicating coupling TAT to these peptides could be considered as a potential tool to improve their antimicrobial activity. Overall, we have shown that targeted modifications of this natural antimicrobial peptide can adjust its bioactivities to help its development as an antibiotic or anti-proliferative agent.

Identification of novel Amurin-2 variants from the skin secretion of Rana amurensis, and the design of cationicity-enhanced analogues.

Rana amurensis is important in Chinese medicine as its skin secretions contain abundant bioactive peptides. Here, we have identified the antimicrobial peptide Amurin-2 and three highly-conserved variants, Amurin-2a, Amurin-2b and Amurin-2c through a combination of molecular cloning and MS/MS fragmentation sequencing. Synthetic replicates of these peptides demonstrate potent antimicrobial activity against S. aureus, whilst some have activity against C.albicans and even resistant bacterial MRSA. Furthermore, two Lys-analogues (K4-Amurin-2 and K11-Amurin-2) were designed to improve the bioactive function and the antimicrobial activity of K4-Amurin-2 against E.coli was enhanced distinctly. In addition, the two modified peptides also showed more potent activity against S. aureus, C. albicans and MRSA strains. Meanwhile, these peptides showed inhibitory effect on the cell viability of several cancer cells. As a result, these structural and functional studies of Amurin-2 variants and analogues could provide insights for future antimicrobial peptide design.

USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells.

BACKGROUND: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs). METHODS: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed. RESULTS: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib. CONCLUSIONS: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

Unlocking the Doors to Patient Satisfaction in Pediatric Orthopaedics.

BACKGROUND: Despite efforts to enhance the patient experience, many health care providers continue to struggle to improve patient satisfaction as the identification of tangible quality improvement areas remains difficult. This dilemma is particularly relevant in pediatric settings, where patient satisfaction measures have not been as thoroughly studied in subspecialties such as orthopaedics. We investigate this issue to identify the major drivers of patient satisfaction in pediatric orthopaedics, which has significant financial and professional implications for both hospital administrators and health care providers. Although recent patient experience studies emphasize on improving access to care and nurturing hospitality by facilities upgrades or staff development, we hypothesized that the patient-physician relationship remains the most important factor in patients' assessment of their experiences. METHODS: Patient satisfaction surveys were collected from outpatient visits to pediatric orthopaedic practices at 5 locations in 3 states. Data were aggregated as monthly percentages of responses on a 5-point Likert scale. Month over month Pearson product-moment correlation coefficients were generated between top responses for "Likelihood of Your Recommending Our Practice to Others" (LTR) and other variables. RESULTS: In total, 6195 families completed satisfaction surveys. The variables most predictive of likelihood to recommend the practice were "Staff Worked Together" (r=0.82), "Friendliness/Courtesy of Care Provider" (r=0.80), "Cheerfulness of Practice" (r=0.80), "Likelihood of Recommending Care Provider" (r=0.80), and "Care Provider's Information about Medications" (r=0.78). CONCLUSIONS: Measurements of the patient-physician relationship, along with overall cheerfulness and staff collaboration, have the strongest relationships to LTR. These results suggest that patient satisfaction is influenced by more than just the patient-physician relationship, and may have significance in aiding pediatric orthopaedic clinics in their quality assurance/quality improvement plans of enhancing the patient experience. LEVEL OF EVIDENCE: Level III-prognostic.

Deubiquitylating enzymes in receptor endocytosis and trafficking.

In recent times, our knowledge of the roles ubiquitin plays in multiple cellular processes has expanded exponentially, with one example being the role of ubiquitin in receptor endocytosis and trafficking. This has prompted a multitude of studies examining how the different machinery involved in the addition and removal of ubiquitin can influence this process. Multiple deubiquitylating enzymes (DUBs) have been implicated either in facilitating receptor endocytosis and lysosomal degradation or in rescuing receptor levels by preventing endocytosis and/or promoting recycling to the plasma membrane. In this review, we will discuss in detail what is currently known about the role of DUBs in regulating the endocytosis of various transmembrane receptors and ion channels. We will also expand upon the role DUBs play in receptor sorting at the multivesicular body to determine whether a receptor is recycled or trafficked to the lysosome for degradation. Finally, we will briefly discuss how the DUBs implicated in these processes may contribute to the pathogenesis of a range of diseases, and thus the potential these have as therapeutic targets.

Evaluation of patient satisfaction in pediatric dermatology.

BACKGROUND/OBJECTIVES: There remains a lack of investigation into which factors patients and families value the most in their experience at pediatric dermatology clinics. Most of the current literature on quality improvement in dermatology does not encompass the pediatric population. To determine the drivers that are most predictive of a positive patient experience, we observed the indirect relationship between several factors of the patient experience and their role in patient satisfaction. METHODS: Patient satisfaction surveys were distributed after their visits to patients at four pediatric dermatology clinics in one children's academic health system. Data were collected and organized into the top 30 survey variables with which patients expressed satisfaction on a 5-point Likert scale. Pearson product-moment correlation coefficients (r) for each variable with regard to "likelihood of your recommending our practice to others" were calculated. RESULTS: A total of 516 families completed patient satisfaction surveys. Analyses of top box scores showed that the strongest predictors of patient satisfaction were the likelihood of recommending care provider (r = .77, P = <.001), cheerfulness of practice (r = .76, P = <.001), care provider spoke using clear language (r = .73, P = <.001), patient confidence in care provider (r = .70, P = <.001), and our sensitivity to patient needs (r = .70, P = <.001). CONCLUSION: The patient-physician relationship, along with the environment of the practice and its sensitivity to patients' personal needs, contributes most to the patient experience in pediatric dermatology. Identifying such variables that shape patients' assessments of their experience can guide future quality improvement plans in the specialty.

Key drivers of patient experience in ambulatory paediatric cardiology.

Patient experience is becoming a central focus of healthcare. A broad range of studies on how to increase patient satisfaction ratings exists; however, they lack the specificity to adequately guide physicians and hospitals on how to improve patient experience. The objective of this study was to define the aspects of patient experience within paediatric cardiologist practices that can serve as predictors of excellent patient satisfaction. From 1 January, 2013 to 28 February, 2015 (26 months), outpatients who visited paediatric cardiologists were asked to complete a 39-question patient satisfaction survey regarding their experience. Surveys were collected over a 26-month period by Press Ganey, an independent provider of patient satisfaction surveys. Participants were asked to rate their experience on a 1-5 Likert-scale: a score of 1 demonstrated a "poor" experience, whereas a score of 5 demonstrated a "very good" experience. This retrospective study of 2468 responses determined that cheerfulness of the practice (r=0.85, p<0.001), a cohesive staff (r=0.83, p<0.001), and a care provider explaining problems and conditions (r=0.81, p<0.001) were key aspects of a paediatric cardiologist's practice that can be used as predictors of overall patient satisfaction. Awareness of how doctors can personalise a patient's experience is vital to achieve greater patient satisfaction and, ultimately, better patient outcomes.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages.

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation. METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1ß response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover. RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1ß release from macrophages, through lysosomal destabilization. IL-1ß secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1ß release was significantly reduced in primary macrophages from ctss(-/-) mice. CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1ß release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

A novel RCE1 isoform is required for H-Ras plasma membrane localization and is regulated by USP17.

Processing of the 'CaaX' motif found on the C-termini of many proteins, including the proto-oncogene Ras, requires the ER (endoplasmic reticulum)-resident protease RCE1 (Ras-converting enzyme 1) and is necessary for the proper localization and function of many of these 'CaaX' proteins. In the present paper, we report that several mammalian species have a novel isoform (isoform 2) of RCE1 resulting from an alternate splice site and producing an N-terminally truncated protein. We demonstrate that both RCE1 isoform 1 and the newly identified isoform 2 are required to reinstate proper H-Ras processing and thus plasma membrane localization in RCE1-null cells. In addition, we show that the deubiquitinating enzyme USP17 (ubiquitin-specific protease 17), previously shown to modulate RCE1 activity, can regulate the abundance and localization of isoform 2. Furthermore, we show that isoform 2 is ubiquitinated on Lys43 and deubiquitinated by USP17. Collectively, the findings of the present study indicate that RCE1 isoform 2 is required for proper 'CaaX' processing and that USP17 can regulate this via its modulation of RCE1 isoform 2 ubiquitination.

Optimising intracellular delivery of antibiotic loaded PLGA nanoparticles to macrophages.

Bacteria can evade antimicrobial therapy by hiding inside host cells such as macrophages. Here we examine the ability of PLGA nanoparticles to deliver antibiotics to intracellular bacteria, specifically focusing upon the impact of nanoparticle size. Different sized Rhodamine-B conjugated PLGA nanoparticles were synthesized and uptake examined in two macrophage cell lines, as well as different epithelial cells, to determine the optimal properties for macrophage uptake. These studies demonstrate macrophages display a consistent increase in uptake with increased PLGA nanoparticle diameter. In a bacteria-macrophage co-culture model, we then examined the efficacy of different sized antibiotic-loaded PLGA nanoparticles against intracellular infections with K. pneumoniae and S. aureus. Increasing the size of antibiotic-loaded PLGA nanoparticles significantly increased their potency against intracellular K. pneumoniae. However, this was not observed for S. aureus, potentially due to the observation these nanoparticles failed to access the compartment in which S. aureus reside. This work demonstrates for the first time that increasing the size of antibiotic-loaded PLGA nanoparticles can significantly enhance antimicrobial efficacy against K. pneumoniae intracellular macrophage infections. However, our S. aureus studies indicate this is not a 'one size fits all' approach for all intracellular infections.

An Effective Modification Strategy to Build Multifunctional Peptides Based on a Trypsin Inhibitory Peptide of the Kunitz Family.

Peptides with antimicrobial activity or protease inhibitory activity are potential candidates to supplement traditional antibiotics or cancer chemotherapies. However, the potential of many peptides are limited by drawbacks such as cytotoxicity or susceptibility to hydrolysis. Therefore, strategies to modify the structure of promising peptides may represent an effective approach for developing more promising clinical candidates. In this study, the mature peptide OSTI-1949, a Kunitz-type inhibitor from Odorrana schmackeri, and four designed analogues were successfully synthesised. In contrast to the parent peptide, the analogues showed impressive multi-functionality including antimicrobial, anticancer, and trypsin inhibitory activities. In terms of safety, there were no obvious changes observed in the haemolytic activity at the highest tested concentration, and the analogue OSTI-2461 showed an increase in activity against cancer cell lines without cytotoxicity to normal cells (HaCaT). In summary, through structural modification of a natural Kunitz-type peptide, the biological activity of analogues was improved whilst retaining low cytotoxicity. The strategy of helicity enhancement by forming an artificial α-helix and ß-sheet structure provides a promising way to develop original bioactive peptides for clinical therapeutics.

Cathepsin S from both tumor and tumor-associated cells promote cancer growth and neovascularization.

Recent murine studies have demonstrated that tumor-associated macrophages in the tumor microenvironment are a key source of the pro-tumorigenic cysteine protease, cathepsin S. We now show in a syngeneic colorectal carcinoma murine model that both tumor and tumor-associated cells contribute cathepsin S to promote neovascularization and tumor growth. Cathepsin S depleted and control colorectal MC38 tumor cell lines were propagated in both wild type C57Bl/6 and cathepsin S null mice to provide stratified depletion of the protease from either the tumor, tumor-associated host cells, or both. Parallel analysis of these conditions showed that deletion of cathepsin S inhibited tumor growth and development, and revealed a clear contribution of both tumor and tumor-associated cell derived cathepsin S. The most significant impact on tumor development was obtained when the protease was depleted from both sources. Further characterization revealed that the loss of cathepsin S led to impaired tumor vascularization, which was complemented by a reduction in proliferation and increased apoptosis, consistent with reduced tumor growth. Analysis of cell types showed that in addition to the tumor cells, tumor-associated macrophages and endothelial cells can produce cathepsin S within the microenvironment. Taken together, these findings clearly highlight a manner by which tumor-associated cells can positively contribute to developing tumors and highlight cathepsin S as a therapeutic target in cancer.

Enhanced antitumor activity of the photosensitizer meso-Tetra(N-methyl-4-pyridyl) porphine tetra tosylate through encapsulation in antibody-targeted chitosan/alginate nanoparticles.

meso-Tetra(N-methyl-4-pyridyl) porphine tetra tosylate (TMP) is a photosensitizer that can be used in photodynamic therapy (PDT) to induce cell death through generation of reactive oxygen species in targeted tumor cells. However, TMP is highly hydrophilic, and therefore, its ability to accumulate intracellularly is limited. In this study, a strategy to improve TMP uptake into cells has been investigated by encapsulating the compound in a hydrogel-based chitosan/alginate nanoparticle formulation. Nanoparticles of 560 nm in diameter entrapping 9.1 µg of TMP per mg of formulation were produced and examined in cell-based assays. These particles were endocytosed into human colorectal carcinoma HCT116 cells and elicited a more potent photocytotoxic effect than free drug. Antibodies targeting death receptor 5 (DR5), a cell surface apoptosis-inducing receptor up-regulated in various types of cancer and found on HCT116 cells, were then conjugated onto the particles. The conjugated antibodies further enhanced uptake and cytotoxic potency of the nanoparticle. Taken together, these results show that antibody-conjugated chitosan/alginate nanoparticles significantly enhanced the therapeutic effectiveness of entrapped TMP. This novel approach provides a strategy for providing targeted site-specific delivery of TMP and other photosensitizer drugs to treat colorectal tumors using PDT.

Cathepsin V regulates cell cycle progression and histone stability in the nucleus of breast cancer cells.

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of the molecular role of the protease within Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome. Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation by MTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment. Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells. Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation.

Development of a cationic polyethyleneimine-poly(lactic-co-glycolic acid) nanoparticle system for enhanced intracellular delivery of biologics.

Intracellular delivery of proteins, peptides and biologics is an emerging field which has the potential to provide novel opportunities to target intracellular proteins, previously deemed 'undruggable'. However, the delivery of proteins intracellularly remains a challenge. Here, we present a cationic nanoparticle delivery system for enhanced cellular delivery of proteins through use of a polyethyleneimine and poly-(lactic-co-glycolic acid) polymer blend. Cationic nanoparticles were shown to provide increased cellular uptake compared to anionic and neutral nanoparticles, successfully delivering Variable New Antigen Receptors (vNARs), entrapped within the nanoparticle core, to the cell interior. vNARs were identified as ideal candidates for nanoparticle entrapment due to their remarkable stability. The optimised 10% PEI-PLGA nanoparticle formulation displayed low toxicity, was uniform in size and possessed appropriate cationic charge to limit cellular toxicity, whilst being capable of escaping the endo/lysosomal system and delivering their cargo to the cytosol. This work demonstrates the ability of cationic nanoparticles to facilitate intracellular delivery of vNARs, novel biologic agents with potential utility towards intracellular targets.