The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer.

The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi's sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.

Variability in the prevalence of depression among adults with chronic pain: UK Biobank analysis through clinical prediction models.

BACKGROUND: The prevalence of depression among people with chronic pain remains unclear due to the heterogeneity of study samples and definitions of depression. We aimed to identify sources of variation in the prevalence of depression among people with chronic pain and generate clinical prediction models to estimate the probability of depression among individuals with chronic pain. METHODS: Participants were from the UK Biobank. The primary outcome was a "lifetime" history of depression. The model's performance was evaluated using discrimination (optimism-corrected C statistic) and calibration (calibration plot). RESULTS: Analyses included 24,405 patients with chronic pain (mean age 64.1 years). Among participants with chronic widespread pain, the prevalence of having a "lifetime" history of depression was 45.7% and varied (25.0-66.7%) depending on patient characteristics. The final clinical prediction model (optimism-corrected C statistic: 0.66; good calibration on the calibration plot) included age, BMI, smoking status, physical activity, socioeconomic status, gender, history of asthma, history of heart failure, and history of peripheral artery disease. Among participants with chronic regional pain, the prevalence of having a "lifetime" history of depression was 30.2% and varied (21.4-70.6%) depending on patient characteristics. The final clinical prediction model (optimism-corrected C statistic: 0.65; good calibration on the calibration plot) included age, gender, nature of pain, smoking status, regular opioid use, history of asthma, pain location that bothers you most, and BMI. CONCLUSIONS: There was substantial variability in the prevalence of depression among patients with chronic pain. Clinically relevant factors were selected to develop prediction models. Clinicians can use these models to assess patients' treatment needs. These predictors are convenient to collect during daily practice, making it easy for busy clinicians to use them.

Human Papillomavirus 16 E2 Interaction with TopBP1 Is Required for E2 and Viral Genome Stability during the Viral Life Cycle.

CK2 phosphorylation of HPV16 E2 at serine 23 promotes interaction with TopBP1, and this interaction is important for E2 plasmid segregation function. Here, we demonstrate that the E2-TopBP1 interaction is critical for E2 and viral genome stability during the viral life cycle. Introduction of the S23A mutation into the HPV16 genome results in a loss of E2 expression and viral genome integration during organotypic rafting. Coculture of N/Tert-1+E2-S23A cells with J2 fibroblasts results in E2-S23A degradation via the proteasome; wild-type E2 is not degraded. TopBP1 siRNA treatment of N/Tert-1+E2-WT cells results in E2 degradation only in the presence of J2 cells demonstrating the critical role for TopBP1 in maintaining E2 stability. The CK2 inhibitor CX4945 promotes E2-WT degradation in the presence of fibroblasts as it disrupts E2-TopBP1 interaction. siRNA targeting SIRT1 rescues E2-S23A stability in N/Tert-1 cells treated with J2 fibroblasts, with an increased E2-S23A acetylation. The results demonstrate that the E2-TopBP1 interaction is critical during the viral life cycle as it prevents fibroblast stimulated SIRT1 mediated deacetylation of E2 that promotes protein degradation. This means that the E2-TopBP1 complex maintains E2 and viral genome stability and that disruption of this complex can promote viral genome integration. Finally, we demonstrate that HPV11 E2 also interacts with TopBP1 and that this interaction is critical for HPV11 E2 stability in the presence of J2 cells. Treatment of N/Tert-1 + 11E2-WT cells with CX4945 results in 11E2 degradation. Therefore, CK2 inhibition is a therapeutic strategy for alleviating HPV11 diseases, including juvenile respiratory papillomatosis. IMPORTANCE Human papillomaviruses are pathogens that cause a host of diseases ranging from benign warts to cancers. There are no therapeutics available for combating these diseases that directly target viral proteins or processes; therefore, we must enhance our understanding of HPV life cycles to assist with identifying novel treatments. In this report, we demonstrate that HPV16 and HPV11 E2 protein expression is dependent upon TopBP1 interaction in keratinocytes interacting with fibroblasts, which recapitulate stromal interactions in culture. The degradation of 16E2 promotes HPV16 genome integration; therefore, the E2-TopBP1 interaction is critical during the viral life cycle. We demonstrate that the CK2 inhibitor CX4945 disrupts HPV11 interaction with TopBP1 and destabilizes HPV11 E2 protein in the presence of J2 fibroblasts; we propose that CX4945 could alleviate HPV11 disease burden.

Direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for human papillomavirus 16 E2 association with mitotic chromatin and plasmid segregation function.

IMPORTANCE: Human papillomavirus 16 (HPV16) is a causative agent in around 3%-4% of all human cancers, and currently, there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.

Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis.

Human papillomavirus 16 (HPV16) E2 is a DNA-binding protein that regulates transcription, replication and potentially, segregation of the HPV16 genome during the viral life cycle. In the segregation model, E2 simultaneously binds to viral and host chromatin, acting as a bridge to ensure that viral genomes reside in daughter nuclei following cell division. The host chromatin receptor for E2 mediating this function is unknown. Recently, we demonstrated that CK2 phosphorylation of E2 on serine 23 (S23) is required for interaction with TopBP1, and that this interaction promotes E2 and TopBP1 recruitment to mitotic chromatin. Here, we demonstrate that in U2OS cells expressing wild-type E2 and a non-TopBP1-binding mutant (S23A, serine 23 mutated to alanine), interaction with TopBP1 is essential for E2 recruitment of plasmids to mitotic chromatin. Using novel quantitative segregation assays, we demonstrate that interaction with TopBP1 is required for E2 plasmid segregation function in U2OS and N/Tert-1 cells. Small interfering RNA (siRNA) knockdown of TopBP1 or CK2 enzyme components disrupts E2 segregation/retention function. The interaction of E2 with TopBP1 promotes increased levels of E2 protein during mitosis in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes (HFK) immortalized by the HPV16 genome. Overall, our results demonstrate that E2 has plasmid segregation activity, and that the E2-TopBP1 interaction is essential for this E2 function. IMPORTANCE HPV16 causes 3% to 4% of all human cancers. It is proposed that during the viral life cycle, the viral genome is actively segregated into daughter nuclei, ensuring viral replication in the subsequent S phase. The E2 protein potentially bridges the viral and host genomes during mitosis to mediate segregation of the circular viral plasmid. Here, we demonstrate that E2 has the ability to mediate plasmid segregation, and that this function is dependent upon interaction with the host protein TopBP1. Additionally, we demonstrate that the E2-TopBP1 interaction promotes enhanced E2 expression during mitosis, which likely promotes the plasmid segregation function of E2. Overall, our results present a mechanism of how HPV16 can segregate its viral genome during an active infection, a critical aspect of the viral life cycle.

Identifying patterns of neurocognitive dysfunction through direct comparison of children with leukemia, central nervous system tumors, and sickle cell disease.

PURPOSE: To quantify and compare the magnitude and type of neurocognitive dysfunction in at-risk children with central nervous system (CNS) tumors, acute lymphoblastic leukemia (ALL), and sickle cell disease (SCD) using a common instrument and metric to directly compare these groups with each other. METHODS: Fifty-three participants between the ages of 7 and 12 years (n = 27 ALL, n = 11 CNS tumor, n = 15 SCD) were enrolled and assessed using the NIH Toolbox Cognition Battery (NIHTCB). Participants with ALL or CNS tumor were 0-18 months posttherapy, while participants with SCD possessed the SS or Sß0 genotype, took hydroxyurea, and had no known history of stroke. RESULTS: Independent sample t-tests showed that participants with ALL and CNS tumor experienced greatest deficits in processing speed (ALL d = -0.96; CNS tumor d = -1.2) and inhibitory control and attention (ALL d = -0.53; CNS tumor d = -0.97) when compared with NIHTCB normative data. Participants with SCD experienced deficits in cognitive flexibility only (d = -0.53). Episodic memory was relatively spared in all groups (d = -0.03 to -0.32). There were no significant differences in function when groups were compared directly with each other by analysis of variance. CONCLUSIONS: Use of a common metric to quantify the magnitude and type of neurocognitive dysfunction across at-risk groups of participants by disease shows that participants perform below age-expected norms in multiple domains and experience dysfunction differently than one another. This approach highlights patterns of dysfunction that can inform disease- and domain-specific interventions.

Optimal cut-offs of depression screening tools during the COVID-19 pandemic: a systematic review.

BACKGROUND: Studies have reported an increase in the prevalence of depression during the COVID-19 pandemic. The accuracy of screening tools may change with the prevalence and distribution of a disease in a population or sample: the "Spectrum Effect". METHODS: First, we selected commonly used screening tools and developed search strategies for the inclusion of original studies during the pandemic. Second, we searched PsycINFO, EMBASE, and MEDLINE from March 2020 to September 2022 to obtain original studies that investigated the accuracy of depression screening tools during the pandemic. We then searched these databases to identify meta-analyses summarizing the accuracy of these tools conducted before the pandemic and compared the optimal cut-offs for depression screening tools during the pandemic with those before. RESULT: Four original studies evaluating the optimal cut-offs for four screening tools (Beck Depression Inventory [BDI-II], Hospital Anxiety and Depression Scale-Depression [HADS-D], Patient Health Questionnaire-9 [PHQ-9], and Geriatric Depression Scale-4 [GDS-4]) were published during the pandemic. Four meta-analyses summarizing these tools before the pandemic. We found that the optimal cut-off of BDI-II was 14 during the pandemic (23.8% depression prevalence, screening patients with Type 2 diabetes) and 14.5 before the pandemic (17.6% depression prevalence, screening psychiatric, primary care, and healthy populations); HADS-D was 10 during the pandemic (23.8% depression prevalence, screening patients with type 2 diabetes) and 7 before the pandemic (15.0% depression prevalence, screening medically ill patients); PHQ-9 was 11 during the pandemic (14.5% depression prevalence, screening university students) and 8 before the pandemic (10.9% depression prevalence, screening the unrestricted population), and GDS-4 was 1.8 during the pandemic (29.0% depression prevalence, screening adults seen in a memory clinic setting) and 3 before the pandemic (18.5% depression prevalence, screening older adults). CONCLUSION: The optimal cut-off for different screening tools may be sensitive to changes in study populations and reference standards. And potential spectrum effects that should be considered in post-COVID time which aiming to improve diagnostic accuracy.

Engagement in Prescription Opioid Tapering Research: the EMPOWER Study and a Coproduction Model of Success.

Patients with chronic pain experience stigma within the healthcare system. This stigma is compounded for those taking long-term prescription opioids. Often, public messaging and organizational policies have telegraphed that opioid treatment is a problem to be solved by focusing only on medication reduction efforts. Lack of data has contributed to misperceptions and poor opioid policies. In part, data collection remains poor because patients feel fractured from systems of care and are often not interested in engaging with opioid reduction mandates and research. Similarly, clinicians may fail to engage with opioid stewardship and research due to complexities that exceed their training or capacities. The EMPOWER study applies a coproduction model that engages researchers, patients, clinicians, managers, and other health system users. Key stakeholders shaped the design of the study to best ensure acceptability and engagement of the "end users"-patients who enroll in the study and the clinicians who implement the opioid tapers. Targeting the needs of any stakeholder group in isolation is suboptimal. Accordingly, we detail the EMPOWER patient-centered opioid tapering clinical research framework and specific strategies to address stakeholder concerns. We also discuss how this framework may be applied to enhance engagement in healthcare research broadly.

Identifying predictors of HPV-related head and neck squamous cell carcinoma progression and survival through patient-derived models.

Therapeutic innovation for human papilloma virus-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) is impaired by inadequate preclinical models and the absence of accurate biomarkers. Our study establishes the first well-characterized panel of patient-derived xenografts (PDXs) and organoids from HPV+ HNSCCs while determining fidelity of the models to the distinguishing genetic features of this cancer type. Despite low engraftment rates, whole exome sequencing showed that PDXs retain multiple distinguishing features of HPV+ HNSCC lost in existing cell lines, including PIK3CA mutations, TRAF3 deletion and the absence of EGFR amplifications. Engrafted HPV+ tumors frequently contained NOTCH1 mutations, thus providing new models for a negatively prognostic alteration in this disease. Genotype-phenotype associations in the models were then tested for prediction of tumor progression and survival in published clinical cohorts. Observation of high tumor mutational burdens (TMBs) in the faster-growing models facilitated identification of a novel association between TMB and local progression in both HPV+ and HPV- patients that was prognostic in HPV- cases. In addition, reduced E7 and p16INK4A levels found in a PDX from an outlier case with lethal outcome led to detection of similar profiles among recurrent HPV+ HNSCCs. Transcriptional data from the Cancer Genome Atlas was used to demonstrate that the lower E2F target gene expression predicted by reduced E7 levels has potential as a biomarker of disease recurrence risk. Our findings bridge a critical gap in preclinical models for HPV+ HNSCCs and simultaneously reveal novel potential applications of quantifying mutational burden and viral oncogene functions for biomarker development.

Human Papillomavirus 16 E2 Regulates Keratinocyte Gene Expression Relevant to Cancer and the Viral Life Cycle.

Human papillomaviruses (HPVs) are causative agents in ano-genital and oropharyngeal cancers. The virus must reprogram host gene expression to promote infection, and E6 and E7 contribute to this via the targeting of cellular transcription factors, including p53 and pRb, respectively. The HPV16 E2 protein regulates host gene expression in U2OS cells, and in this study, we extend these observations into telomerase reverse transcriptase (TERT) immortalized oral keratinocytes (NOKs) that are capable of supporting late stages of the HPV16 life cycle. We observed repression of innate immune genes by E2 that are also repressed by the intact HPV16 genome in NOKs. Transcriptome sequencing (RNA-seq) data identified 167 up- and 395 downregulated genes by E2; there was a highly significant overlap of the E2-regulated genes with those regulated by the intact HPV16 genome in the same cell type. Small interfering RNA (siRNA) targeting of E2 reversed the repression of E2-targeted genes. The ability of E2 to repress innate immune genes was confirmed in an ano-genital immortalized keratinocyte cell line, N/Tert-1. We present the analysis of data from The Cancer Genome Atlas (TCGA) for HPV16-positive and -negative head and neck cancers (HNC) suggesting that E2 plays a role in the regulation of the host genome in cancers. Patients with HPV16-positive HNC with a loss of E2 expression exhibited a worse clinical outcome, and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.IMPORTANCE Human papillomavirus 16 (HPV16)-positive tumors that retain expression of E2 have a better clinical outcome than those that have lost E2 expression. It has been suggested that this is due to a loss of E2 repression of E6 and E7 expression, but this is not supported by data from tumors where there is not more E6 and E7 expression in the absence of E2. Here we report that E2 regulates host gene expression and place this regulation in the context of the HPV16 life cycle and HPV16-positive head and neck cancers (the majority of which retain E2 expression). We propose that this E2 function may play an important part in the increased response of HPV16-positive cancers to radiation therapy. Therefore, host gene regulation by E2 may be important for promotion of the HPV16 life cycle and also for the response of HPV16-positive tumors to radiation therapy.

Deprescribing long-term opioid therapy in patients with chronic pain.

Proposed regulatory changes will limit the access to opioids by Australian patients with chronic pain, many of whom are under the care of consultant physicians. This review summarises points of consensus on opioid deprescribing that emerged from the interaction of an expert panel and the audience at a symposium on the topic held in Sydney in 2019. Each of these consensus points speaks to the need for an individualised, patient-centred approach. In other words, 'treat the patient, not the pill count'.

Can orthognathic surgery be expected to improve patients' psychological well-being? The challenge of hedonic adaptation.

In addition to improved oral health and function, many people are motivated to undergo orthognathic surgery to improve their facial aesthetics and overall psychological well-being (daily affect and satisfaction with life). This article explains the phenomenon of hedonic adaptation, which challenges the notion that patients treated with orthognathic surgery can expect to experience improvements in psychological well-being following surgery. We review evidence for hedonic adaptation across a variety of life circumstances and explain the psychological processes which account for the relative stability of people's well-being over their life course despite significant positive (and negative) changes in their circumstances. We examine the conditions that assist and inhibit hedonic adaptation to positive life events, and argue that if patients are made aware of these processes, they are more likely to achieve sustained improvements in psychological well-being following orthognathic surgery. Based on our narrative review of evidence for hedonic adaptation, we conclude that orthognathic surgery should not be expected to improve patients' psychological well-being in the long term and suggest that patients be advised to take intentional steps to preserve any improvements in their day-to-day affect and satisfaction with life that arise as a result of their surgery.

The human papillomavirus type 16 L1 protein directly interacts with E2 and enhances E2-dependent replication and transcription activation.

The human papillomavirus (HPV) E2 protein is a multifunctional protein essential for the control of virus gene expression, genome replication and persistence. E2 is expressed throughout the differentiation-dependent virus life cycle and is functionally regulated by association with multiple viral and cellular proteins. Here, we show for the first time to our knowledge that HPV16 E2 directly associates with the major capsid protein L1, independently of other viral or cellular proteins. We have mapped the L1 binding region within E2 and show that the α-2 helices within the E2 DNA-binding domain mediate L1 interaction. Using cell-based assays, we show that co-expression of L1 and E2 results in enhanced transcription and virus origin-dependent DNA replication. Upon co-expression in keratinocytes, L1 reduces nucleolar association of E2 protein, and when co-expressed with E1 and E2, L1 is partially recruited to viral replication factories. Furthermore, co-distribution of E2 and L1 was detected in the nuclei of upper suprabasal cells in stratified epithelia of HPV16 genome-containing primary human keratinocytes. Taken together, our findings suggest that the interaction between E2 and L1 is important for the regulation of E2 function during the late events of the HPV life cycle.

The role of protein kinase A regulation of the E6 PDZ-binding domain during the differentiation-dependent life cycle of human papillomavirus type 18.

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

HPV16 genome structure analysis in oropharyngeal cancer PDXs identifies tumors with integrated and episomal genomes.

HPV + oropharyngeal squamous cell carcinoma (OPC) incidence recently surpassed cervical cancer and is the most common HPV-related cancer in the developed world. HPV16 is in ∼90 % of HPV + OPCs, with episomal genomes in the majority of cases. Most existing HPV16+ cancer cell lines derive from outside the oropharynx and harbor integrated HPV genomes. Thus, there is need for OPC preclinical models to evaluate standard and experimental therapeutics in the presence of episomal HPV16 oncogenic drivers. Here we characterize HPV genome structures in eight HPV16+ OPC patient-derived xenografts (PDXs), and evaluate their responses to standard chemotherapy. HPV genome state was investigated by combining Southern blot, T5 exonuclease assay, whole genome sequencing, and RNAseq data. This analysis revealed complexity and variation in integrated vs. episomal HPV forms across PDXs and demonstrated that four PDXs predominantly contain episomal HPV16. Episomal status did not ensure favorable in vivo responses to cisplatin therapy, despite the more favorable prognosis previously attributed to episomal HPV + tumors; this could be due to the small number present in the dataset. Our analysis establishes PDX models as test platforms for novel therapies designed to target maintenance of the episomal forms of HPV16 that commonly appear in OPC.

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We also demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. The results present a model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis and E2 acetylation on K111 by p300 increases, promoting interaction with Top1 that protects K112 from ubiquitination and therefore E2 proteasomal degradation. Importance: Human papillomaviruses are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

Inhibition of Human Cytomegalovirus Entry into Mucosal Epithelial Cells.

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

Impact of healthcare interventions on distress following acute musculoskeletal/orthopaedic injury: a scoping review of systematic reviews.

BACKGROUND: Musculoskeletal injuries can cause distress, and distress is associated with delayed recovery. Numerous interventions have been developed to facilitate recovery from injury, and several systematic reviews evaluate the efficacy of these interventions for reducing psychological distress. OBJECTIVES: This scoping review aims to map the synthesised evidence for the relationship between treatment interventions and distress-related outcomes following acute injury. The objectives were (1) to describe the types of interventions that have been evaluated in relation to distress-related outcomes following accidental injury, (2) to examine the scope of distress-related outcomes that have been measured in relation to these interventions and (3) to explore the range of clinical professions that deliver these interventions. DESIGN: We searched nine electronic databases and grey literature (to 21 April 2022). We included any systematic review reporting on the relationship between interventions delivered in the time following injury and distress-related outcomes. Data relevant to the specific objectives of this scoping review were extracted and described using narrative synthesis. RESULTS: From 8412 systematic reviews imported for screening, 8266 unique records were screened. 179 were selected for full-text review. 84 systematic reviews were included in the study. Interventional types were pharmacological, psychological, exercise based, physical/manual therapies, virtual reality based, multimodal and workplace based. Interventions were delivered digitally, face to face and using virtual reality by a variety of healthcare professionals, including doctors, nurses, psychologists and physiotherapists. The most frequently reported distress-related variables included anxiety, depression, post-traumatic stress disorder diagnosis. CONCLUSION: A wide range of interventions may help to mitigate distress following acute accidental musculoskeletal or orthopaedic injury. Even interventions that were not designed to reduce distress were found to improve distress-related outcomes. In view of the important role of distress in recovery from injury, it is recommended that distress-related variables are measured as core outcomes in the evaluation of treatments for acute injuries.