Identifying regulatory elements and their RNA-binding proteins in the 3' untranslated regions of papillomavirus late mRNAs.

Human papillomaviruses (HPVs) infect cutaneous and mucosal epithelia to cause benign (warts) and malignant lesions (e.g. cervical cancer). Bovine papillomaviruses (BPVs) infect fibroblasts to cause fibropapillomas but can also infect cutaneous epithelial cells. For HPV-1, -16, -31 and BPV-1, cis-acting RNA elements in the late 3' untranslated region (3'UTR) control expression of virus proteins by binding host cell proteins. The present study compared the effects on gene expression of the cis-acting elements of seven PV late 3'UTRs (HPV-6b, -11, -16, -31 and BPV-1, -3 and -4) representing a range of different genera and species and pathological properties. pSV-beta-galactosidase reporter plasmids containing the late 3'UTRs from seven PVs were transiently transfected into cervical adenocarcinoma HeLa cells, and reporter gene expression quantified by reverse transcription-quantitative PCR and a beta-galactosidase assay. All elements inhibited gene expression in keratinocytes. Cancer-related types HPV-16 and -31, had the greatest inhibitory activity whereas the lowest inhibition was found in the non-cancer related types, BPV-3 and HPV-11. Using RBPmap version 1.1, bioinformatics predictions of factors binding the elements identified proteins which function mainly in mRNA splicing. Markedly, in terms of protein binding motifs, BPV late 3'UTR elements were similar to those of HPV-1a but not to other HPVs. Using HPV-1a as a model and siRNA depletion, the bioinformatics predictions were tested and it was found that PABPC4 was responsible for some of the 3'UTR repressive activity. The data revealed candidate proteins that could control PV late gene expression.

The human papillomavirus late life cycle and links to keratinocyte differentiation.

Regulation of human papillomavirus (HPV) gene expression is tightly linked to differentiation of the keratinocytes the virus infects. HPV late gene expression is confined to the cells in the upper layers of the epithelium where the virus capsid proteins are synthesized. As these proteins are highly immunogenic, and the upper epithelium is an immune-privileged site, this spatial restriction aids immune evasion. Many decades of work have contributed to the current understanding of how this restriction occurs at a molecular level. This review will examine what is known about late gene expression in HPV-infected lesions and will dissect the intricacies of late gene regulation. Future directions for novel antiviral approaches will be highlighted.

Microwave hyperthermia represses human papillomavirus oncoprotein activity and induces cell death due to cell stress in 3D tissue models of anogenital precancers and cancers.

BACKGROUND: Hyperthermia is a well-accepted cancer therapy. Microwaves provide a very precise, targeted means of hyperthermia and are currently used to treat plantar warts caused by cutaneous-infective human papillomaviruses (HPVs). Other HPV genotypes infecting the anogenital mucosa cause genital warts or preneoplastic lesions or cervical cancer. Effective, non-ablative therapies for these morbid HPV-associated lesions are lacking. METHODS: The molecular consequences of microwave treatment were investigated in in vitro cultured three-dimensional HPV-positive cervical tumour tissues, and tissues formed from HPV-infected normal immortalised keratinocytes. Microwave energy delivery to tissues was quantified. Quantitative reverse transcriptase PCR was used to quantify mRNA expression. Immunohistochemistry and fluorescence immunostaining was used to assess protein expression. FINDINGS: Microwave energy deposition induced sustained, localised cell death at the treatment site. There was a downregulation in levels of HPV oncoproteins E6 and E7 alongside a reduction in cellular growth/proliferation and induction of apoptosis/autophagy. HSP70 expression confirmed hyperthermia, concomitant with induction of translational stress. INTERPRETATION: The data suggest that microwave treatment inhibits tumour cell proliferation and allows the natural apoptosis of HPV-infected cells to resume. Precision microwave delivery presents a potential new treatment for treating HPV-positive anogenital precancerous lesions and cancers. FUNDING: Funding was through an Innovate UK Biomedical Catalyst grant (ID# 92138-556187), a Chief Scientist Office grant (TCS/19/11) and core support from Medical Research Council (MC_ UU_12014) core funding for the MRC-University of Glasgow Centre for Virus Research.

HPV status and HPV16 viral load in anal cancer and its association with clinical outcome.

BACKGROUND: The incidence of anal cancer is increasing globally. Evidence-based improvement in early detection and management of this morbid cancer is thus required. In other cancers associated with Human Papillomavirus (HPV), viral status and dynamics, including viral load (VL) has been shown to influence clinical outcome. Our aim was to determine the influence of HPV status and HPV16 VL on the clinical outcomes of anal cancer patients. METHODS: A total of 185 anal cancer lesions were genotyped for HPV. Of the HPV16 positive component, VL was determined using a digital droplet PCR assay. The association of qualitative HPV status and VL (low (<12.3), medium (12.3-57) and high (>57 copies/cell)) on overall survival and hazard of death was assessed. RESULTS: Of the 185 cases, 164 (88.6%) samples were HPV positive. HPV16 was detected in 154/185 samples (83.2%). HPV positive status was associated with improved overall survival in the univariate analysis [hazard ratio (HR) of 0.44, 0.23-0.82, p = 0.01]. When adjusted by age, sex, stage and response to treatment, the association of positive HPV status with improved survival remained (HR 0.24 [0.11-0.55] p < 0.001). High VL was associated with improved overall survival in the univariate analysis with a HR of 0.28 (0.11-0.71, p = 0.007). When adjusted only by age and sex, high VL was associated with better overall survival (HR 0.27, 0.11-0.68 p = 0.006). CONCLUSIONS: HPV status appears to be independently associated with improved outcomes in anal cancer patients. Moreover, HPV viral load quantification may be informative for further risk stratification and warrants further investigation.

High occurrence of postintensive care syndrome identified in surgical ICU survivors after implementation of a multidisciplinary clinic.

BACKGROUND: Postintensive care syndrome (PICS) has been identified in a large proportion of medical intensive care unit survivors; however, the occurrence surgical intensive care unit (SICU) survivors is unknown. We implemented a multidisciplinary critical care outpatient clinic (CCOC) to identify the occurrence of PICS in SICU survivors. METHODS: Seventy acute care surgery and trauma patients, 18 years or older, who remained in the SICU for 72 hours or longer at a Level I trauma center were seen in CCOC at 2 weeks, 12 weeks, and 24 weeks after hospital discharge. The CCOC staffing included a nurse coordinator, social worker, critical care pharmacist, physical therapist, and acute care surgeon who identified PICS sequelae in their respective specialties by clinical criteria and screening questionnaires. RESULTS: Of 82 eligible patients, 70 (85.4%) were seen at least once for 116 total visits. Forty-three (61.4%) patients suffered traumatic injuries and 27 (38.6%) underwent emergent general surgery. Sixty-seven (95.7%) demonstrated at least one PICS criterion. Over all visits, 26 (37.1%) patients presented with one PICS criterion, 24 (34.3%) patients with two, and 17 (24.3%) with three. Cognitive impairment was observed in 29 (41.4%) patients, psychiatric in 30 (42.9%), and physical symptoms in 65 (92.9%). Activity Measure for Post-Acute Care scores improved from severe impairment at admission to full function by 12 weeks postdischarge, yet 6 Minute Walk Test scores remained below age-matched references through all visits. Patients expressed mild to moderate depression based on Patient Health Questionnaire-9 scores. A medication reconciliation was completed at 96.5% (112/116) of the visits with 116 total medication recommendations. By 24 weeks following discharge, only 26.4% (14/53) of previously employed patients had resumed work. CONCLUSION: Through the successful implementation of a multidisciplinary CCOC, this study identifies an exorbitant rate of PICS among SICU survivors. LEVEL OF EVIDENCE: Therapeutic/epidemiological, level III.

Contagious itching is heightened in children with autism spectrum disorders.

The authors tested susceptibility to contagious itching, laughter, and yawning in 55 children with autism spectrum disorder (ASD), ages 8-14, and 106 typically developing (TD) children, ages 5-14. Children with ASD were less likely to yawn or laugh contagiously compared with TD peers, but showed increased susceptibility to contagious itching, under naturalistic conditions. Contagious yawning and laughter were positively correlated with emotional empathy in the TD group. In contrast, contagious itching showed no relationship to empathy, and was positively correlated with autism symptom severity in the ASD group. The authors explore the implications of these findings in terms of psychological theories about ASD.

Restraint Reduction, Restraint Elimination, and Best Practice: Role of the Clinical Nurse Specialist in Patient Safety.

PURPOSE: Baseline restraint prevalence for surgical step-down unit was 5.08%, and for surgical intensive care unit, it was 25.93%, greater than the National Database of Nursing Quality Indicators (NDNQI) mean. Project goal was sustained restraint reduction below the NDNQI mean and maintaining patient safety. BACKGROUND/RATIONALE: Soft wrist restraints are utilized for falls reduction and preventing device removal but are not universally effective and may put patients at risk of injury. Decreasing use of restrictive devices enhances patient safety and decreases risk of injury. DESCRIPTION: Phase 1 consisted of advanced practice nurse-facilitated restraint rounds on each restrained patient including multidisciplinary assessment and critical thinking with bedside clinicians including reevaluation for treatable causes of agitation and restraint indications. Phase 2 evaluated less restrictive mitts, padded belts, and elbow splint devices. Following a 4-month trial, phase 3 expanded the restraint initiative including critical care requiring education and collaboration among advanced practice nurses, physician team members, and nurse champions. EVALUATION AND OUTCOMES: Phase 1 decreased surgical step-down unit restraint prevalence from 5.08% to 3.57%. Phase 2 decreased restraint prevalence from 3.57% to 1.67%, less than the NDNQI mean. Phase 3 expansion in surgical intensive care units resulted in wrist restraint prevalence from 18.19% to 7.12% within the first year, maintained less than the NDNQI benchmarks while preserving patient safety. INTERPRETATION/CONCLUSION: The initiative produced sustained reduction in acute/critical care well below the NDNQI mean without corresponding increase in patient medical device removal. IMPLICATIONS: By managing causes of agitation, need for restraints is decreased, protecting patients from injury and increasing patient satisfaction. Follow-up research may explore patient experiences with and without restrictive device use.

How influences on teenage smoking reflect gender and society in Mali, West Africa.

OBJECTIVES: To provide further understanding and discussion on the influences on smoking in young people in Mali. DESIGN: A generic qualitative methodological approach was used following Caelli's generic principles. Six focus group discussions were conducted with a total of 31 participants followed by two semi-structured interviews. A reflexive account was kept to record development in the researcher's theoretical position SETTING: The setting was recreational areas of Bamako, capital city of Mali, West Africa. PARTICIPANTS: Participants aged 13-15 years were recruited opportunistically in a recreational area of Bamako. MAINOUTCOME MEASURES: To develop further understanding of the influences of teenage smoking in Mali, West Africa. RESULTS: FIVE MAIN CATEGORIES THAT EXPLAINED INFLUENCES ON YOUTH SMOKING EMERGED: knowledge and awareness of smoking; associations with smoking; influential people; key messages in Malian society; and access to tobacco. The results showed that influences were complex and interwoven, notable gender differences were revealed, and the role of elder members of the community proved decisive in participants' smoking experiences. Participants described vague knowledge of the impact on health of smoking and reported trying smoking from an early age. Often contact with smoking was through elders and being sent to buy and sometimes light cigarettes for them. Associations with smoking were influenced by gender with smoking more desirable for boys than girls. CONCLUSIONS: Any approach to preventing smoking initiation in young people requires an understanding of the social influences and pressures on young people. A tobacco control strategy is required to look at all areas of influence on smoking behaviours. Different needs should also account for the differing characteristics and perceptions of specific population groups.