Interaction of the Papillomavirus E2 Protein with Mitotic Chromosomes

AbstractThe bovine papillomavirus E2 transactivator protein is a multifunctional protein that activates viral transcription, cooperates in initiation of viral DNA replication, and is required for long-term episomal maintenance of viral genomes. We have shown previously that the E2 transactivator protein and bovine papillomavirus type 1 genomes are associated with mitotic chromosomes and have proposed that E2 links the genomes to cellular chromosomes to ensure segregation to daughter nuclei. In this study, we show that E2 is associated with cellular chromosomes at all stages of mitosis. We also further map the regions of E2 that are required for this association. The transactivation domain of E2 is necessary and sufficient to mediate the interaction with mitotic chromosomes; the DNA binding domain, and the flexible hinge region that separates the two domains, is not required. Furthermore, mutation of previously identified phosphorylation sites (serine residues 235, 298, and 301) has no effect on the ability of the E2 protein to bind mitotic chromosomes

The Transactivation and DNA Binding Domains of the BPV-1 E2 Protein Have Different Roles in Cooperative Origin Binding with the E1 Protein

AbstractThe bovine papillomavirus E2 transactivator protein enhances the ability of the E1 protein to bind to the viral origin of replication which contains an E1 binding site flanked by two E2 binding sites. To determine which regions and functions of the E2 protein are important for this cooperative interaction, a series of mutated E2 proteins were assayed for their ability to enhance E1 origin-specific binding. Cooperative origin binding required at least one E2 DNA binding site, an intact functional E2 DNA binding domain, and an intact transactivation domain. The hinge region of the E2 proteins was dispensable for this activity. To further examine the role of the E2 C-terminal domain, a series of chimeric proteins were generated that substituted the yeast GAL4 DNA binding domain for the E2 DNA binding domain. These chimeric proteins were able to cooperatively bind to a hybrid origin that contained GAL4 binding sites in place of the E2 binding sites. These studies indicate that the E2 transactivation domain is sufficient for interaction with the E1 protein and that the E2 DNA binding domain is required for interaction with origin DNA sequences

The Epstein-Barr Virus Episome Maneuvers between Nuclear Chromatin Compartments during Reactivation.

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription

Host cell restriction factors that limit transcription and replication of human papillomavirus

The life cycle of human papillomaviruses (HPV) is tightly regulated by the differentiation state of mucosal and cutaneous keratinocytes. To counteract viral infection, constitutively expressed cellular factors, which are defined herein as restriction factors, directly mitigate viral gene expression and replication. In turn, some HPV gene products target these restriction factors and abrogate their anti-viral effects to establish efficient gene expression and replication programs. Ironically, in certain circumstances, this delicate counterbalance between viral gene products and restriction factors facilitates persistent infection by HPVs. This review serves to recapitulate the current knowledge of nuclear restriction factors that directly affect the HPV infectious cycle

Paying for Unapproved Medical Products

This symposium article examines the use of investigational (un-approved) medical products in the United States, with particular focus on who pays for this use. In the United States, the question of who pays for the use of approved medical products for their intended indications is complicated enough, with some expenses borne by private payers, some by public payers, some covered as charity care, and some paid out of pocket by patients. A separate question is off-label use, in which an approved medical product is used for an unapproved indication. In this article, we focus on a narrower issue: what entities in the United States pay for access to unapproved medical products, e.g., investigational drugs, devices, or diagnostics that have not (yet) received Food and Drug Administration (“FDA”) approval. We examine the various forms of preapproval access (“PAA”) to experimental medical products available in the United States—clinical trials and non-trial preapproval access via the Expanded Access (“EA”) and Right to Try (“RTT”) pathways. For each, this paper analyzes which entity—individual, insurer, sponsor, or other—bears the cost and what limitations or caps, if any, exist on these costs. This paper considers various proposed novel payment mechanisms that may permit more equitable use of investigational medical products. Part I outlines payment-related disparities in access in- grained in the current United States healthcare system. Part II focuses on access in the context of clinical trials, which most payers have begun to cover, but where remaining uncovered expenses can disincentivize participation in clinical trials, even among those highly motivated to enroll. Part III discusses non-trial preapproval access pathways, specifically Expanded Access and Right to Try, where coverage is scant. Part IV briefly deals with investigational products (such as stem cell treatments) that are available via unregulated or underregulated direct-to-consumer sales. Part V then re- views the ethical considerations inherent in paying for investigational medical products

Investigating the impact of financial concerns on symptoms of depression in UK healthcare workers: data from the UK-REACH nationwide cohort study

BACKGROUND: Exploration of the association between financial concerns and depression in UK healthcare workers (HCWs) is paramount given the current 'cost of living crisis', ongoing strike action and recruitment/retention problems in the National Health Service. AIMS: To assess the impact of financial concerns on the risk of depression in HCWs, how these concerns have changed over time and what factors might predict financial concerns. METHOD: We used longitudinal survey data from a UK-wide cohort of HCWs to determine whether financial concerns at baseline (December 2020 to March 2021) were associated with depression (measured with the Public Health Questionnaire-2) at follow-up (June to October 2022). We used logistic regression to examine the association between financial concerns and depression, and ordinal logistic regression to establish predictors of developing financial concerns. RESULTS: A total of 3521 HCWs were included. Those concerned about their financial situation at baseline had higher odds of developing depressive symptoms at follow-up. Financial concerns increased in 43.8% of HCWs and decreased in 9%. Those in nursing, midwifery and other nursing roles had over twice the odds of developing financial concerns compared with those in medical roles. CONCLUSIONS: Financial concerns are increasing in prevalence and predict the later development of depressive symptoms in UK HCWs. Those in nursing, midwifery and other allied nursing roles may have been disproportionately affected. Our results are concerning given the potential effects on sickness absence and staff retention. Policy makers should act to alleviate financial concerns to reduce the impact this may have on a discontent workforce plagued by understaffing

Productivity costs from a dengue episode in Asia: a systematic literature review

Background Dengue is a mosquito-borne viral infection which has been estimated to cause a global economic burden of US$8.9 billion per year. 40 Method We searched PubMed and Web of Knowledge without date and language restrictions using terms related to dengue and cost and economics burden. The titles and abstracts of publications related to Asia were screened to identify relevant studies. The reported productivity losses and costs of non-fatal and fatal dengue episodes were then described and compared. Costs were adjusted for inflation to 2017 prices. Results We reviewed 33 relevant articles, of which 20 studies reported the productivity losses, and 31 studies reported productivity costs. The productivity costs varied between US$6.7–1445.9 and US$3.8–1332 for hospitalized and outpatient non-fatal episodes, respectively. The productivity cost associated with fatal dengue episodes varied between US$12,035-1,453,237. A large degree of this variation was due to the range of different countries being investigated and their corresponding economic status. However, estimates for a given country still showed notable variation. Conclusion We found that the estimated productivity costs associated with dengue episodes in Asia are notable. However, owing to the significant variation in methodology and approaches applied, the reported productivity costs of dengue episodes were often not directly comparable across studies. More consistent and transparent methodology regarding the estimation of productivity costs would help the estimates of the economic burden of dengue be more accurate and comparable across studies

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

Stroke Ready: a multi-level program that combines implementation science and community-based participatory research approaches to increase acute stroke treatment: protocol for a stepped wedge trial

Abstract Background Post-stroke disability is common, costly, and projected to increase. Acute stroke treatments can substantially reduce post-stroke disability, but few patients take advantage of these cost-effective treatments. Practical, cost-efficient, and sustainable interventions to address underutilized acute stroke treatments are currently lacking. In this context, we present the Stroke Ready project, a stepped wedge design, multi-level intervention that combines implementation science and community-based participatory research approaches to increase acute stroke treatments in the predominately African American community of Flint, Michigan, USA. Methods Guided by the Tailored Implementation of Chronic Disease (TICD) framework, we begin with optimization of acute stroke care in emergency departments, with particular attention given to our safety-net hospital partners. Then, we move to a community-wide, multi-faceted, stroke preparedness intervention, with workshops led by peer educators, over 2 years. Measures of engagement of the safety-net hospital and the feasibility and sustainability of the implementation strategy as well as community intervention reach, dose delivered, and satisfaction will be collected. The primary outcome is acute stroke treatment rates, which includes both intravenous tissue plasminogen activator, and endovascular treatment. The co-secondary outcomes are intravenous tissue plasminogen activator treatment rates and the proportion of stroke patients who arrive by ambulance. Discussion If successful, Stroke Ready will increase acute stroke treatment rates through emergency department and community level interventions. The stepped wedge design and process evaluation will provide insight into how Stroke Ready works and where it might work best. By exploring the relative effectiveness of the emergency department optimization and the community intervention, we will inform hospitals and communities as they determine how best to use their resources to optimize acute stroke care. Trial registration ClinicalTrials.gov Trial Identifier NCT03645590 .https://deepblue.lib.umich.edu/bitstream/2027.42/148211/1/13012_2019_Article_869.pd