A Grounded Theory Exploration of Addictions Treatment Within a Commercial Airline Setting.

INTRODUCTION: An Australasian Airline's Alcohol and Other Drug (AOD) Program demonstrates abstinence rates that exceed those of general AOD programs. The reasons for this are unclear. The purpose of this research was to develop a theory as to why this program is successful.METHODS: A qualitative examination following grounded theory methodology was undertaken. AOD program patients and healthcare professionals were interviewed until content saturation was reached. Data analysis followed grounded theory to identify the key concepts associated with the program's success.RESULTS: The core theory that emerged highlighted the pivotal roles of a strong employee-company relationship, shared values, and a safety-focused culture in explicating the program's success. This moves beyond the "carrot and stick" model of motivation, where belonging to this organization and safety consciousness serve as powerful drivers for abstinence. Challenges and barriers highlighted some unique challenges to the program in managing the coronavirus pandemic and the difference in approach to substance use in community spaces versus safety-critical employment.DISCUSSION: This research expands the understanding of this AOD program's success in a safety-critical industry, emphasizing the elements of a working relationship that are beyond positive or negative reinforcement. Future research should work to quantify and test the generalizability of these findings.Nairn J, Bell E, Myers J, Higgins M, Johnston B, Newton-Howes G. A grounded theory exploration of addictions treatment within a commercial airline setting. Aerosp Med Hum Perform. 2024; 95(6):313-320.

Virion-associated US28 rapidly modulates Akt activity to suppress HCMV lytic replication in monocytes.

Establishing a non-productive quiescent/silent infection within monocytes is essential for spread of human cytomegalovirus (HCMV). Yet, how HCMV establishes a quiescent infection in monocytes remains unclear. US28 is a viral G protein-coupled receptor (GPCR) essential for silent infections within cells of the myeloid lineage. We found virion-associated US28 was rapidly delivered to monocytes, while de novo synthesized US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28Δ) was unable to establish a quiescent infection, resulting in a fully productive lytic replication cycle. Mechanistically, viral entry of US28Δ phosphorylated Akt at both serine 473 (S473) and threonine 308 (T308), which contrasted with the site-specific phosphorylation of Akt at S473 following WT infection. Preventing Akt bi-phosphorylation prevented lytic replication of US28Δ, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of WT infection. Our data demonstrate that virion-delivered US28 fine-tunes Akt activity to permit HCMV infection to enter a quiescent state following primary infection of monocytes.

Retinoblastoma Protein Is Required for Epstein-Barr Virus Replication in Differentiated Epithelia.

Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.

Incidence, Prevalence, and Sources of COVID-19 Infection among Healthcare Workers in Hospitals in Malaysia.

The COVID-19 pandemic introduced significant novel risks for healthcare workers and healthcare services. This study aimed to determine the prevalence, trends, characteristics, and sources of COVID-19 infection among healthcare workers during the early COVID-19 pandemic in Malaysian hospitals. A cross-sectional study used secondary data collected from a COVID-19 surveillance system for healthcare workers between January and December 2020. Two surges in COVID-19 cases among healthcare workers in Malaysia were epidemiologically correlated to a similarly intense COVID-19 pattern of transmission in the community. The period prevalence of COVID-19 infection and the mortality rate among healthcare workers in Malaysia were 1.03% and 0.0019%, respectively. The majority of infections originated from the workplace (53.3%); a total of 36.3% occurred among staff; a total of 17.0% occurred between patients and staff; and 43.2% originated from the community. Healthcare workers had a 2.9 times higher incidence risk ratio for the acquisition of COVID-19 infection than the general population. Nursing professionals were the most highly infected occupational group (40.5%), followed by medical doctors and specialists (24.1%), and healthcare assistants (9.7%). The top three departments registering COVID-19 infections were the medical department (23.3%), the emergency department (17.7%), and hospital administration and governance (9.1%). Occupational safety and health units need to be vigilant for the early detection of a disease outbreak to prevent the avoidable spread of disease in high-risk settings. The transformation of some tertiary hospitals to dedicated COVID-19 care, the monitoring of new procedures for the management of COVID-19 patients, and appropriate resource allocation are key to successful risk mitigation strategies.

Protocol for assessing ex vivo lipolysis of murine adipose tissue.

Here, we provide a detailed protocol for assessing ex vivo lipolysis of subcutaneous and visceral white adipose tissue. We describe a robust approach to detect depot-specific changes in lipolytic potential under basal and beta-adrenergic receptor-stimulated conditions. Given that adipose tissue plays a critical role in systemic metabolic health, this experimental protocol can be used to determine changes in adipose tissue function in health and disease.

Harnessing cortical plasticity via gabapentinoid administration promotes recovery after stroke.

Stroke causes devastating sensory-motor deficits and long-term disability due to disruption of descending motor pathways. Restoration of these functions enables independent living and therefore represents a high priority for those afflicted by stroke. Here, we report that daily administration of gabapentin, a clinically approved drug already used to treat various neurological disorders, promotes structural and functional plasticity of the corticospinal pathway after photothrombotic cortical stroke in adult mice. We found that gabapentin administration had no effects on vascular occlusion, haemodynamic changes nor survival of corticospinal neurons within the ipsilateral sensory-motor cortex in the acute stages of stroke. Instead, using a combination of tract tracing, electrical stimulation and functional connectivity mapping, we demonstrated that corticospinal axons originating from the contralateral side of the brain in mice administered gabapentin extend numerous collaterals, form new synaptic contacts and better integrate within spinal circuits that control forelimb muscles. Not only does gabapentin daily administration promote neuroplasticity, but it also dampens maladaptive plasticity by reducing the excitability of spinal motor circuitry. In turn, mice administered gabapentin starting 1 h or 1 day after stroke recovered skilled upper extremity function. Functional recovery persists even after stopping the treatment at 6 weeks following a stroke. Finally, chemogenetic silencing of cortical projections originating from the contralateral side of the brain transiently abrogated recovery in mice administered gabapentin, further supporting the conclusion that gabapentin-dependent reorganization of spared cortical pathways drives functional recovery after stroke. These observations highlight the strong potential for repurposing gabapentinoids as a promising treatment strategy for stroke repair.

Integrative Review of Non-Technical Skills Frameworks to Apply for Air Medical Transfer of Pregnant Women.

OBJECTIVE: To identify the most suitable non-technical skills framework to adapt and apply to the air medical transfer of pregnant women. DATA SOURCES: Embase, PsycINFO, PubMed, MEDLINE, Web of Science, CINAHL, Science Direct, and Google Scholar. STUDY SELECTION: We retrieved potentially relevant articles using a predefined combination of keywords extended with truncation and Boolean operators. Database and manual reference searches yielded 569 peer-reviewed articles. We included articles if they presented empirical data and described non-technical or cognitive competency skills frameworks for health care professionals. We discussed any ambiguities regarding inclusion, and they were resolved by consensus. We retained 71 full-text articles for final review. DATA EXTRACTION: We coded extracted data under four criteria: non-technical skill categories, context of use, psychometric properties, and rating system. We generated descriptive summary tables of the characteristics of existing non-technical skills frameworks based on publication year, method of development, clinical setting, clinical specialty, routine/crisis-based performance, and team/individual performance. DATA SYNTHESIS: We identified 42 non-technical skills frameworks from a variety of health care settings. We critically examined context of use and how use in various clinical settings may align with air transfers of pregnant women. Our findings illustrate the importance of team-based and routine performance rather than crisis-focused skills. Maintaining situational awareness throughout all stages of the transfer and communicating effectively with team members, the pregnant woman, and her partner are skills that are particularly important to ensure good outcomes. CONCLUSION: We selected the Global Assessment of Obstetric Team Performance as the most suitable non-technical skills framework to adapt to the clinical setting of air medical transfer of pregnant women. We considered the clinical specialty, specific non-technical skills required in the setting, the framework's properties, and the requirement to focus on routine team performance.

Missed cervical spine injuries: aim for the top.

PURPOSE: To determine the incidence of missed cervical spine injuries by radiology registrars in a major trauma centre and to identify any common blind spots. MATERIALS AND METHODS: All patients with an acute traumatic injury who underwent a CT scan of the cervical spine in our unit, which serves a population of approximately 900,000, between September 2016 and December 2017 and whom had a separate radiology trainee report and final neuroradiology consultant report available were included in the study. We recorded the date and time of the scan, the registrar error and the registrar grade. An error was defined as follows: (1) a missed fracture; (2) a missed ligamentous injury; (3) overcall of a fracture (e.g. degenerative calcification or nutrient vessel). Groups were compared with the chi-square test. RESULTS: Five hundred seventy-three CT scans of the cervical spine fitted the inclusion criteria and were analysed. There were a total of 149 injuries over eight levels in 96 patients. There were 12 registrar errors (2.1% discrepancy rate), of which 11 were missed acute injuries (9 fractures and 2 disco-ligamentous injuries). The grade of the registrar was not significant (p = 0.603). Seventy-three percent (8/11) missed injuries were disproportionately at the cranio-cervical junction, where only 11.6% of traumatic cervical spine injuries occur p < 0.0001. Forty-five percent of the missed injuries included occipital condyle fractures, which occurred in only 12/149 injuries (8%). CONCLUSIONS: Radiology registrars safely report emergency CT scans of the cervical spine performed following trauma with a low discrepancy rate. Missed cervical spine injuries commonly occur at the cranio-cervical junction, which should become a standard review area.

Keeping it going: the importance of delivering interprofessional education during the COVID-19 pandemic.

BACKGROUND AND CONTEXT Globally, the coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for better interprofessional collaboration and teamwork. When disciplines have worked together to undertake testing, deliver care and administer vaccines, progress against COVID-19 has been made. Yet, teamwork has often not happened, wasting precious resources and stretching health-care workforces. Continuing to train health professionals during the pandemic is challenging, particularly delivering interprofessional education that often uses face-to-face delivery methods to optimise interactional learning. Yet, continuing to offer interprofessional education throughout the pandemic is critical to ensure a collaboration-ready health workforce. One example is continuing the established INVOLVE (Interprofessional Visits to Learn Interprofessional Values through Patient Experience) interprofessional education initiative. ASSESSMENT OF PROBLEM Educators have not always prioritised interprofessional education during the pandemic, despite its immediate and long-term benefits. The INVOLVE interprofessional education initiative, usually delivered face-to-face, was at risk of cancellation. RESULTS A quality improvement analysis of the strategies used to continue INVOLVE demonstrated that it is possible to deliver interprofessional education within the constraints of a pandemic by using innovative online and hybrid educational strategies. Educators and students demonstrated flexibility in responding to the sudden changes in teaching and learning modalities. STRATEGIES When pandemic alert levels change, interprofessional educators and administrators can now choose from a repertoire of teaching approaches. LESSONS Four key lessons have improved the performance and resilience of INVOLVE: hold the vision to continue interprofessional education; be nimble; use technology appropriately; and there will be silver linings and unexpected benefits to the changes.

Butyrate-producing human gut symbiont, Clostridium butyricum, and its role in health and disease.

Clostridium butyricum is a butyrate-producing human gut symbiont that has been safely used as a probiotic for decades. C. butyricum strains have been investigated for potential protective or ameliorative effects in a wide range of human diseases, including gut-acquired infection, intestinal injury, irritable bowel syndrome, inflammatory bowel disease, neurodegenerative disease, metabolic disease, and colorectal cancer. In this review we summarize the studies on C. butyricum supplementation with special attention to proposed mechanisms for the associated health benefits and the supporting experimental evidence. These mechanisms center on molecular signals (especially butyrate) as well as immunological signals in the digestive system that cascade well beyond the gut to the liver, adipose tissue, brain, and more. The safety of probiotic C. butyricum strains appears well-established. We identify areas where additional human randomized controlled trials would provide valuable further data related to the strains' utility as an intervention.

Genomic Diversity of Burkholderia pseudomallei Isolates, Colombia.

We report an analysis of the genomic diversity of isolates of Burkholderia pseudomallei, the cause of melioidosis, recovered in Colombia from routine surveillance during 2016-2017. B. pseudomallei appears genetically diverse, suggesting it is well established and has spread across the region.

Housing Interventions for Intimate Partner Violence Survivors: A Systematic Review.

Intimate partner violence (IPV) survivors are much more likely to experience housing insecurity or homelessness than those who have not experienced IPV. However, little comprehensive research has evaluated the effectiveness of interventions used to address IPV survivors' housing insecurity. To address this knowledge gap, our team conducted a systematic review guided by three questions: (a) What are current interventions for addressing IPV survivors' housing needs? (b) What are the methodological strengths and limitations of the research evaluating those interventions? (c) How effective are the identified interventions? We identified potentially relevant peer-reviewed and gray literature using variations of predetermined search terms and four search methods. Twelve articles met inclusion criteria. Accordingly, this study showed that there is an overall dearth of research concerning interventions that address IPV survivors' housing insecurity and needs. Shelter is the most commonly assessed and available housing intervention for IPV survivors, but only limited empirical evaluation is available of shelter effectiveness. In addition, findings indicate both traditional shelter services and innovative interventions (e.g., rapid rehousing, flexible funding) would benefit from rigorous evaluation including examining survivor and situation characteristics contributing to housing strategy effectiveness.

An Exonuclease V-qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues.

Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC. Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues Alternate Protocol: Determining HPV integration type or integrity of HPV episome.

Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis.

BACKGROUND: Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strains in the susceptible BALB/c mouse model. METHODOLOGY/PRINCIPAL FINDING: In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. CONCLUSIONS/SIGNIFICANCE: Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies.

Suppression of Stromal Interferon Signaling by Human Papillomavirus 16.

Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs) and HFKs containing either wild-type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Interferon (IFN)-stimulated genes (ISGs) and extracellular matrix remodeling genes were suppressed, the most prominent pathways affected. STAT1, IFNAR1, IRF3, and IRF7 were knocked down in stromal fibroblasts using lentiviral short hairpin RNA (shRNA) transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated with increased late keratinocyte differentiation but not decreased IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation.IMPORTANCE The persistence of high-risk human papillomavirus (HPV) infections is the key risk factor for developing HPV-associated cancers. The ability of HPV to evade host immunity is a critical component of its ability to persist. The environment surrounding a tumor is increasingly understood to be critical in cancer development, including immune evasion. Our studies show that HPV can suppress the expression of immune-related genes in neighboring fibroblasts in a three-dimensional (3D) model of human epithelium. This finding is significant, because it indicates that HPV can control innate immunity not only in the infected cell but also in the microenvironment. In addition, the ability of HPV to regulate stromal gene expression depends in part on the viral oncogene E5, revealing a new function for this protein as an immune evasion factor.

Burkholderia pseudomallei ΔtonB Δhcp1 Live Attenuated Vaccine Strain Elicits Full Protective Immunity against Aerosolized Melioidosis Infection.

Burkholderia pseudomallei is a Gram-negative facultative intracellular bacterium and the causative agent of melioidosis, a severe infectious disease found throughout the tropics. This organism is closely related to Burkholderia mallei, the etiological agent of glanders disease which primarily affects equines. These two pathogenic bacteria are classified as Tier 1 select agents due to their amenability to aerosolization, limited treatment options, and lack of an effective vaccine. We have previously successfully demonstrated the immunogenicity and protective efficacy of a live attenuated vaccine strain, B. malleiΔtonB Δhcp1 (CLH001). Thus, we applied this successful approach to the development of a similar vaccine against melioidosis by constructing the B. pseudomalleiΔtonB Δhcp1 (PBK001) strain. C57BL/6 mice were vaccinated intranasally with the live attenuated PBK001 strain and then challenged with wild-type B. pseudomallei K96243 by the aerosol route. Immunization with strain PBK001 resulted in full protection (100% survival) against acute aerosolized melioidosis with very low bacterial burden as observed in the lungs, livers, and spleens of immunized mice. PBK001 vaccination induced strong production of B. pseudomallei-specific serum IgG antibodies and both Th1 and Th17 CD4+ T cell responses. Further, humoral immunity appeared to be essential for vaccine-induced protection, whereas CD4+ and CD8+ T cells played a less direct immune role. Overall, PBK001 was shown to be an effective attenuated vaccine strain that activates a robust immune response and offers full protection against aerosol infection with B. pseudomalleiIMPORTANCE In recent years, an increasing number of melioidosis cases have been reported in several regions where melioidosis is endemic and in areas where melioidosis had not commonly been diagnosed. Currently, the estimated burden of disease is around 165,000 new cases annually, including 89,000 cases that have fatal outcomes. This life-threatening infectious disease is caused by B. pseudomallei, which is classified as a Tier 1 select agent. Due to the high case fatality rate, intrinsic resistance to multiple antibiotic treatments, susceptibility to infection via the aerosol route, and potential use as a bioweapon, we have developed an effective live attenuated PBK001 vaccine capable of protecting against aerosolized melioidosis.

Evaluating New Compounds to Treat Burkholderia pseudomallei Infections.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease that requires long-term treatment regimens with no assurance of bacterial clearance. Clinical isolates are intrinsically resistant to most antibiotics and in recent years, isolates have been collected that display resistance to frontline drugs. With the expanding global burden of B. pseudomallei, there is a need to identify new compounds or improve current treatments to reduce risk of relapse. Using the Pathogen Box generated by Medicines for Malaria Venture, we screened a library of 400 compounds for bacteriostatic or bactericidal activity against B. pseudomallei K96243 and identified seven compounds that exhibited inhibitory effects. New compounds found to have function against B. pseudomallei were auranofin, rifampicin, miltefosine, MMV688179, and MMV688271. An additional two compounds currently used to treat melioidosis, doxycycline and levofloxacin, were also identified in the screen. We determined that the minimal inhibitory concentrations (MIC) for levofloxacin, doxycycline, and MMV688271 were below 12 µg/ml for 5 strains of B. pseudomallei. To assess persister frequency, bacteria were exposed to 100x MIC of each compound. Auranofin, MMV688179, and MMV688271 reduced the bacterial population to an average of 4.53 × 10-6% compared to ceftazidime, which corresponds to 25.1% survival. Overall, our data demonstrates that auranofin, MMV688197, and MMV688271 have the potential to become repurposed drugs for treating melioidosis infections and the first evidence that alternative therapeutics can reduce B. pseudomallei persistence.

A new cell culture model to genetically dissect the complete human papillomavirus life cycle.

Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

Characterization of the Burkholderia cenocepacia TonB Mutant as a Potential Live Attenuated Vaccine.

Burkholderia cenocepacia is an opportunistic pathogen prevalent in cystic fibrosis patients, which is particularly difficult to treat, causing chronic and eventually fatal infections. The lack of effective treatment options makes evident the need to develop alternative therapeutic or prophylactic approaches. Vaccines, and live attenuated vaccines, are an unexplored avenue to treat B. cenocepacia infections. Here we constructed and characterized a B. cenocepacia tonB mutant strain, which was unable to actively transport iron, to test whether this single gene deletion mutant (strain renamed GAP001) protected against an acute respiratory B. cenocepacia lethal infection. Here we show that the mutant strain GAP001 is attenuated, and effective at protecting against B. cenocepacia challenge. Intranasal administration of GAP001 to BALB/c mice resulted in almost complete survival with high degree of bacterial clearance.