In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. IMPORTANCE Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection is a risk factor for cancer development and is partly achieved by the attachment of viral DNA to cellular chromatin during cell division. The HPV E2 protein plays a critical role in this tethering by binding simultaneously to the viral genome and to chromatin during mitosis. We previously showed that the cellular DNA helicase ChlR1 is required for loading of the bovine papillomavirus E2 protein onto chromatin during DNA synthesis. Here we identify a mutation in HPV16 E2 that abrogates interaction with ChlR1, and we show that ChlR1 regulates the chromatin association of HPV16 E2 and that this virus-host interaction is essential for viral episome maintenance.

Steroid-induced hyperglycaemia (SIH) is a common adverse effect in patients both with and without diabetes. This project aimed to improve the screening and diagnosis of SIH by improving the knowledge of healthcare professionals who contribute to the management of SIH in hospitalised patients. Monitoring and diagnosis of SIH were measured in areas of high steroid use in our hospital from May 2016 to January 2017. Several interventions were implemented to improve knowledge and screening for SIH including a staff education programme for nurses, healthcare assistants and doctors. The Trust guidelines for SIH management were updated based on feedback from staff. The changes to the guideline included shortening the document from 14 to 4 pages, incorporating a flowchart summarising the management of SIH and publishing the guideline on the Trust intranet. A questionnaire based on the recommendations of the Joint British Diabetes Societies for SIH was used to assess the change in knowledge pre-intervention and post-intervention. Results showed an increase in junior doctors’ knowledge of this topic. Although there was an initial improvement in screening for SIH, this returned to near baseline by the end of the study. This study highlights that screening for SIH can be improved by increasing the knowledge of healthcare staff. However, there is a need for ongoing interventions to sustain this change.

The metaphase chromosome spread technique and subsequent analysis of sister chromatid cohesion is used for (clinical) diagnosis of genetic abnormalities that can cause aberrant sister chromatid cohesion. In addition, the technique can be used to assess the contribution of novel genes to the cohesion establishment and maintenance pathways. Cells are swelled in a hypotonic solution and fixed in Carnoy's solution. Samples are then dropped onto glass slides, and the spread chromosomes are stained and visualized by microscopy. Defects in sister chromatid cohesion can be easily assessed using this method, examples of which are given.

AimsTo assess the current knowledge and practice for monitoring and diagnosis of steroid-induced hyperglycaemia (SIH) in hospitalised patients. MethodsThis study was undertaken on six wards with high frequency of steroid use -oncology, respiratory and neurology -at a large tertiary care centre in the West Midlands, UK. Two anonymous 10-question online surveys -one for junior doctors and another for nurses and healthcare auxiliaries -were used to assess knowledge regarding SIH. The questionnaires were based on the recommendations by the Joint British Diabetes Societies for SIH and approved by the diabetes team at our trust following several discussions for appropriateness.To assess current practice, patients prescribed with two or more consecutive days of oral and/or intravenous steroids during May 2016 in the above areas were assessed. Data for age, gender, history of diabetes, steroid dose, type, route and frequency were collected for each patient. The frequency of preevening meal capillary blood glucose (CBG) monitoring and confirmatory lab glucose for patients with CBG ≥11 mmol/L was measured. The data were analysed using Statistical Analysis Software Package version 23 (IBM Corp., New York, USA). ResultsThirty junior doctors and 42 nurses and auxiliaries completed the survey assessing their knowledge. 50% of doctors, nurses and auxiliaries knew the diagnostic criteria for SIH. While 63% doctors knew the target capillary blood glucose (CBG) range once diagnosed, 70% did not know appropriate follow-up for patients diagnosed with SIH post-discharge. 97% of the nurses correctly reported appropriate monitoring frequency for SIH; however, 25% did not know the correct timeframe to check for diagnostic CBG.135 patients (median age 63 years, range 19-95 years; male: female ratio 1.5:1) were included. 19.3% of patients had been previously diagnosed with diabetes. Dexamethasone (51.9%) was the most commonly used steroid. Pre-evening meal CBG was measured in 55.6% of all patients and was 46.8% (51/109) in patients without diabetes, 92.3% (24/26) in patients with diabetes. 21.6% (11/51) of patients without diabetes had a preevening meal CBG ≥11 mmol/L. Of these, 18.2% (2/11) had lab glucose sent for confirmation of high CBG and none had their HbA 1c measured. ConclusionsThere is a need to improve the knowledge about SIH among healthcare professionals. These data indicate that current practice at the centre falls short of achieving the standards of blood glucose monitoring for patients receiving steroid treatment, as set out in the Joint British Diabetes Societies recommendations. ■