Rab11FIP1-deficient mice develop spontaneous inflammation and show increased susceptibility to colon damage.

The small GTPase, Rab11a, regulates vesicle trafficking and cell polarity in epithelial cells through interaction with Rab11 family-interacting proteins (Rab11-FIPs). We hypothesized that deficiency of Rab11-FIP1 would affect mucosal integrity in the intestine. Global Rab11FIP1 knockout (KO) mice were generated by deletion of the second exon. Pathology of intestinal tissues was analyzed by immunostaining of colonic sections and RNA-sequencing of isolated colonic epithelial cells. A low concentration of dextran sodium sulfate (DSS, 2%) was added to drinking water for 5 days, and injury score was compared between Rab11FIP1 KO, Rab11FIP2 KO, and heterozygous littermates. Rab11FIP1 KO mice showed normal fertility and body weight gain. More frequent lymphoid patches and infiltration of macrophages and neutrophils were identified in Rab11FIP1 KO mice before the development of rectal prolapse compared with control mice. The population of trefoil factor 3 (TFF3)-positive goblet cells was significantly lower, and the ratio of proliferative to nonproliferative cells was higher in Rab11FIP1 KO colons. Transcription signatures indicated that Rab11FIP1 deletion downregulated genes that mediate stress tolerance response, whereas genes mediating the response to infection were significantly upregulated, consistent with the inflammatory responses in the steady state. Lack of Rab11FIP1 also resulted in abnormal accumulation of subapical vesicles in colonocytes and the internalization of transmembrane mucin, MUC13, with Rab14. After DSS treatment, Rab11FIP1 KO mice showed greater body weight loss and more severe mucosal damage than those in heterozygous littermates. These findings suggest that Rab11FIP1 is important for cytoprotection mechanisms and for the maintenance of colonic mucosal integrity.NEW & NOTEWORTHY Although Rab11FIP1 is important in membrane trafficking in epithelial cells, the gastrointestinal phenotype of Rab11FIP1 knockout (KO) mice had never been reported. This study demonstrated that Rab11FIP1 loss induces mistrafficking of Rab14 and MUC13 and decreases in colonic goblet cells, resulting in impaired mucosal integrity.

Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

A proteomic approach to identify endosomal cargoes controlling cancer invasiveness.

We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype.

Using a modified Delphi methodology to gain consensus on the use of dressings in chronic wounds management.

OBJECTIVE: Managing chronic wounds is associated with a burden to patients, caregivers, health services and society and there is a lack of clarity regarding the role of dressings in improving outcomes. This study aimed to provide understanding on a range of topics, including: the definition of chronicity in wounds, the burden of illness, clinical outcomes of reducing healing time and the impact of early interventions on clinical and economic outcomes and the role of matrix metalloproteinases (MMPs) in wound healing. METHOD: A systematic review of the literature was carried out on the role of dressings in diabetic foot ulcer (DFU), and venous leg ulcer (VLU) management strategies, their effectiveness, associated resource use/cost, and quality of life (QoL) impact on patients. From this evidence-base statements were written regarding chronicity in wounds, burden of illness, healing time, and the role of MMPs, early interventions and dressings. A modified Delphi methodology involving two iterations of email questionnaires followed by a face-to-face meeting was used to validate the statements, in order to arrive at a consensus for each. Clinical experts were selected, representing nurses, surgeons, podiatrists, academics, and policy experts. RESULTS: In the first round, 38/47 statements reached or exceeded the consensus threshold of 80% and none were rejected. According to the protocol, any statement not confirmed or rejected had to be modified using the comments from participants and resubmitted. In the second round, 5/9 remaining statements were confirmed and none rejected, leaving 4 to discuss at the meeting. All final statements were confirmed with at least 80% consensus. CONCLUSION: This modified Delphi panel sought to gain clarity from clinical experts surrounding the use of dressings in the management of chronic wounds. A full consensus statement was developed to help clinicians and policy makers improve the management of patients with these conditions.

Association of Combined Estrogen-Progestogen and Progestogen-Only Contraceptives with the Development of Cancer.

Combined estrogen-progestogen contraceptives (oral contraceptives or OCs) and progestogen-only contraceptives (POCs) are synthetic steroids that bind to steroid hormone receptors, which are widespread throughout the body. They have a profound effect on cellular physiology. Combined OCs have been classified by the International Agency for Research on Cancer (IARC) as Group 1 carcinogens, but their findings have not been updated recently. In order to update the information and better understand the impact that OCs and POCs have on the risk of development of cancers, a comprehensive literature search was undertaken, focusing on more recently published papers. In agreement with the IARC, the recent literature confirms an increased risk of breast cancer and cervical cancer with the use of OCs. The recent literature also confirms the IARC conclusion that OCs decrease the risk of ovarian and endometrial cancers. However, there is little support from recent studies for the IARC conclusion that OCs decrease the risk of colorectal cancer or increase the risk of liver cancer. For liver cancer, this may be due to the recent studies having been performed in areas where hepatitis is endemic. In one large observational study, POCs also appear to increase the overall risk of developing cancer. OCs and POCs appear to increase the overall risk of cancer when carefully performed studies with the least intrinsic bias are considered. SUMMARY: OCs have been classified as cancer-causing agents, especially leading to increases in breast cancer and cervical cancer. A review of the recent scientific literature was performed to see whether this still appears to be the case. The recent literature supports the cancer-causing role of OCs especially for breast cancer and cervical cancer. Studies also indicate that progesterone-only contraceptives (such as implants and vaginal rings) also can cause cancer. This is especially true for breast cancer and cervical cancer.

Role of slough and biofilm in delaying healing in chronic wounds.

The management of biofilms with maintenance desloughing and antimicrobial therapy is fast becoming the accepted treatment strategy for chronic wounds.

CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer.

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5ß1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell-matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer.

A randomized controlled trial of web-based training to increase activity in children with cerebral palsy.

AIM: To determine the efficacy of web-based training on activity capacity and performance in children with unilateral cerebral palsy (CP). METHOD: In a matched-pairs randomized waitlist controlled trial, independently ambulant children and adolescents with unilateral CP were allocated to receive 30 minutes of training (intervention) 6 days per week, or usual care (waitlist control) for 20 weeks. Activity capacity was assessed using maximal repetitions of functional strength tasks and 6-minute walk test (6MWT); performance using 4-day ActiGraph GT3X+ accelerometer records at baseline and 20 weeks. Data were analysed by intention to treat comparing between groups using hierarchical linear modelling. RESULTS: Participants were n=101, 52 males, mean age 11 years 3 months (SD 2y 4mo). Intervention participants completed a mean 32.4 hours (SD 17.2) of training, associated with significant improvements in functional strength (mean difference 19.3 repetitions; 95% confidence interval [CI] 10.8-27.7; p<0.001) and 6MWT distance (mean difference 38.9m; 95% CI 12.3-51.9; p<0.001) compared with the control group at 20 weeks, although not activity performance (p>0.05). INTERPRETATION: Training was effective at increasing functional strength and walking endurance in independently ambulant children with unilateral CP. This did not translate into improvements in activity performance.

Non-faith-based arguments against physician-assisted suicide and euthanasia.

This article is a complement to "A Template for Non-Religious-Based Discussions Against Euthanasia" by Melissa Harintho, Nathaniel Bloodworth, and E. Wesley Ely which appeared in the February 2015 Linacre Quarterly. Herein we build upon Daniel Sulmasy's opening and closing arguments from the 2014 Intelligence Squared debate on legalizing assisted suicide, supplemented by other non-faith-based arguments and thoughts, providing four nontheistic arguments against physician-assisted suicide and euthanasia: (1) "it offends me"; (2) slippery slope; (3) "pain can be alleviated"; (4) physician integrity and patient trust. Lay Summary: Presented here are four non-religious, reasonable arguments against physician-assisted suicide and euthanasia: (1) "it offends me," suicide devalues human life; (2) slippery slope, the limits on euthanasia gradually erode; (3) "pain can be alleviated," palliative care and modern therapeutics more and more adequately manage pain; (4) physician integrity and patient trust, participating in suicide violates the integrity of the physician and undermines the trust patients place in physicians to heal and not to harm.

Characteristics associated with physical activity among independently ambulant children and adolescents with unilateral cerebral palsy.

AIM: This study aimed to quantify the contribution of physical, personal and environmental characteristics to physical activity among independently ambulant children with unilateral cerebral palsy (CP). METHOD: One-hundred and two children with unilateral CP (52 males, 50 females; 52 right hemiplegia; mean age 11y 3mo, range 8-17y [SD 2y 4mo]) classified at Gross Motor Function Classification System (GMFCS) levels I = 44 and II = 58 participated. Physical activity was measured over 4 days using ActiGraph accelerometers recording as activity counts. GMFCS, functional strength, 6-minute walk test (6MWT), mobility limitations (MobQues28), age, sex, Assessment of Life-Habits recreation domain, Participation and Environment Measure for Children and Youth (PEM-CY) and environmental characteristics were considered for selection in a linear regression model. These served as independent variables which were determined using a backwards selection procedure. RESULTS: Younger age, male sex, increased performance on the 6MWT, and increased participation in the home and community measured using the PEM-CY were significantly associated with activity counts (p<0.001). However, the model fit was somewhat weak (R(2) =0.32), indicating that much of the variation was unexplained. Older age and reduced community participation were associated with high inactivity (p<0.001). INTERPRETATION: Physical activity interventions should primarily target adolescents and females. Walking endurance and participation in the home and community may represent modifiable characteristics to increase physical activity.

Integrity and virtue: The forming of good character.

Moral character is formed by one's actions. The habits, actions, and emotional responses of the person of good character all are united and directed toward the moral and the good. Because human beings are body/soul unities, actions of the body are actions of the self, that is, human beings are self-possessing, self-governing, and self-determining. In order to be of good character, one must know the good, act in morally good ways, and be disposed and inclined toward the good through the development of virtues. Character and action are intertwined so intimately that one's professional duties, or even what is perceived by others as one's duties, cannot override one's conscience without negatively affecting (and changing) one's character. For the physician to be of good character, it is vital that he or she follow his or her conscience in all things: in private life and also in his or her profession, i.e., in the treatment of patients. Lay summary: Character cannot be separated from the person. To be of good character means that one's habits, actions, and emotional responses all are united and directed toward the moral and the good. In this, public actions cannot be separated from private actions. Both sets of actions affect one's character. For example, a physician believes use of contraceptives to be immoral yet prescribes them in the office because he or she feels a duty to provide what the patient asks for, or a pharmacist who believes abortion to be immoral fills prescriptions for the abortifacient RU-486. These public acts affect one's character even if one's private belief is the opposite of the action. They leave traces on one's character. Not only do actions reflect the goodness or badness of one's character, one's actions also change one's character. The more one does an immoral action or recommends an immoral action for others, the more it becomes part of one's character to be the type of person who condones that immoral action. In order to be of good character one must not only know and desire the good, one must also pursue it in both private and public actions. Virtue is an aid in this; it is the act of good character. Growing in the virtues, especially prudence (knowing what to seek and what to avoid) forms good character. What is at stake is the integrity of the person. The physician who believes that use of contraception is immoral must also act in ways that display that belief and avoid actions that promote contraception use by his or her patients.

Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma.

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification.

Optimisation of Sample Preparation from Primary Mouse Tissue to Maintain RNA Integrity for Methods Examining Translational Control.

The protein output of different mRNAs can vary by two orders of magnitude; therefore, it is critical to understand the processes that control gene expression operating at the level of translation. Translatome-wide techniques, such as polysome profiling and ribosome profiling, are key methods for determining the translation rates occurring on specific mRNAs. These techniques are now widely used in cell lines; however, they are underutilised in tissues and cancer models. Ribonuclease (RNase) expression is often found to be higher in complex primary tissues in comparison to cell lines. Methods used to preserve RNA during lysis often use denaturing conditions, which need to be avoided when maintaining the interaction and position of the ribosome with the mRNA is required. Here, we detail the cell lysis conditions that produce high-quality RNA from several different tissues covering a range of endogenous RNase expression levels. We highlight the importance of RNA integrity for accurate determination of the global translation status of the cell as determined by polysome gradients and discuss key aspects to optimise for accurate assessment of the translatome from primary mouse tissue.

Glioblastoma extracellular vesicles influence glial cell hyaluronic acid deposition to promote invasiveness.

Background: Infiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo-, and radiotherapy. A deeper understanding of the mechanisms invoked by GBM to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. Methods: CRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterized EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived ECM to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. Results: GBM cells expressing a p53 mutant (p53R273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. Conclusions: This work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue.

Disparities in Service and Clinical Outcomes in State-Wide Advanced Practice Physiotherapist-Led Services.

This study explored variations in the primary service and clinical outcomes of a state-wide advanced practice physiotherapist-led service embedded in public medical specialist orthopaedic and neurosurgical outpatient services across Queensland, Australia. An audit of the service database over a six-year period was taken from 18 service facilities. The primary service and clinical outcomes were described. Variations in these outcomes between facilities were explored with a regression analysis adjusting for known patient- and service-related characteristics. The findings showed substantial positive impacts of the advanced practice model across all facilities, with 69.4% of patients discharged without a need for medical specialist review (primary service outcome), consistent with 68.9% of patients reporting clinically important improvements in their condition (primary clinical outcome). However, 15 facilities significantly varied from the state average for the primary service outcome (despite only three facilities varying in the primary clinical outcome). While this disparity in the primary service outcomes appears to be influenced by potentially modifiable differences in the service-related processes between facilities, these process differences only explained part of the variation. This study described the subsequent development of a new, more comprehensive set of service evaluation metrics to better inform future service planning.

PINK1 drives production of mtDNA-containing extracellular vesicles to promote invasiveness.

The cystine-glutamate antiporter, xCT, supports a glutathione synthesis program enabling cancer cells to cope with metabolically stressful microenvironments. Up-regulated xCT, in combination with glutaminolysis, leads to increased extracellular glutamate, which promotes invasive behavior by activating metabotropic glutamate receptor 3 (mGluR3). Here we show that activation of mGluR3 in breast cancer cells activates Rab27-dependent release of extracellular vesicles (EVs), which can transfer invasive characteristics to "recipient" tumor cells. These EVs contain mitochondrial DNA (mtDNA), which is packaged via a PINK1-dependent mechanism. We highlight mtDNA as a key EV cargo necessary and sufficient for intercellular transfer of invasive behavior by activating Toll-like receptor 9 in recipient cells, and this involves increased endosomal trafficking of pro-invasive receptors. We propose that an EV-mediated mechanism, through which altered cellular metabolism in one cell influences endosomal trafficking in other cells, is key to generation and dissemination of pro-invasive microenvironments during mammary carcinoma progression.

Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion.

Signals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that hepatocyte growth factor, an activator of MET (an RTK), promotes Rab17- and clathrin-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2's cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

Investigating inequities in cardiovascular care and outcomes for Queensland Aboriginal and Torres Strait Islander people: protocol for a hospital-based retrospective cohort data linkage project.

INTRODUCTION: Cardiovascular disease (CVD) represents a significant burden of disease for Aboriginal and Torres Strait Islander people, a population that continues to experience a lower life expectancy than other Australians. The aim of the Better Cardiac Care Data Linkage project is to describe patient care pathways and to identify disparities in care and health outcomes between Aboriginal and Torres Strait Islander people and other Queensland residents diagnosed with CVD in the state of Queensland. METHODS: This is a population-based retrospective cohort study using linked regional, state and national health and administrative data collections to describe disparities in CVD healthcare in primary and secondary prevention settings and during hospitalisation. The CVD cohort will be identified from the Queensland Hospital Admitted Patient Data Collection for admissions that occurred between 1 July 2010 and 31 June 2016 and will include relevant International Classification of Disease codes for ischaemic heart disease, congestive heart failure, stroke, acute rheumatic fever and rheumatic heart disease. Person-level data will be linked by Data Linkage Queensland and the Australian Institute of Health and Welfare (AIHW) in accordance with ethical and public health approvals to describe the patient journey prior to, during and post the hospital admission. ANALYSIS: This project will focus largely on descriptive epidemiological measures and multivariate analysis of clinical care standards and outcomes for Aboriginal and Torres Strait Islander people compared with other Queenslanders, including identification of risk factors for suboptimal care and change over time. Variation in care pathways and patient outcomes will be compared by Indigenous status, sex, age group, remoteness of residence, year of index hospitalisation and socioeconomic status. Cox models for time-to-event data and mixed models or generalised estimating equations for longitudinal data will be used to measure change over time where temporal effects exist. ETHICS AND DISSEMINATION: Ethical approval has been granted by Human Research Ethics Committees of the Prince Charles Hospital (HREC/15/QPCH/289) and the AIHW (EO2016-1-233). The Northern Territory Department of Health and Menzies School of Health Research have also provided reciprocal ethical approval of the project (HREC 2019-3490). The deidentified results will be summarised in a report and shared with investigators, advisory groups, Queensland Health and key stakeholders. Findings will be disseminated through workshops, conferences and will be published in peer-reviewed journals.

Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors.

BACKGROUND: Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. RESULTS: Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. CONCLUSIONS: Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.

Quantitative in vivo bioluminescence imaging of orthotopic patient-derived glioblastoma xenografts.

Despite extensive research, little progress has been made in glioblastoma therapy, owing in part to a lack of adequate preclinical in vivo models to study this disease. To mitigate this, primary patient-derived cell lines, which maintain their specific stem-like phenotypes, have replaced established glioblastoma cell lines. However, due to heterogenous tumour growth inherent in glioblastoma, the use of primary cells for orthotopic in vivo studies often requires large experimental group sizes. Therefore, when using intracranial patient-derived xenograft (PDX) approaches, it is advantageous to deploy imaging techniques to monitor tumour growth and allow stratification of mice. Here we show that stable expression of near-infrared fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid, direct non-invasive monitoring of tumour development without compromising tumour stemness or tumorigenicity. Moreover, as this approach does not depend on the use of agents like luciferin, which can cause variability due to changing bioavailability, it can be used for quantitative longitudinal monitoring of tumour growth. Notably, we show that this technique also allows quantitative assessment of tumour burden in highly invasive models spreading throughout the brain. Thus, iRFP transduction of primary patient-derived glioblastoma cells is a reliable, cost- and time-effective way to monitor heterogenous orthotopic PDX growth.