The carcinogenic properties of naturally occurring human papillomavirus 16 E6 oncogene variants

Human papillomavirus is the causative agent of the 2nd highest occurring cancer in women, cervical cancer, which is the result of expression of the E6 and E7 oncogenes. Here we challenged the dogma that the oncogenic protein E6 requires co-expression of E7 for malignant transformation of human keratinocytes using common HPV16 E6 variants that are highly associated with cervical cancer: the Asian-American E6 variant (AAE6) and L83V variant of HPV16. The E6 variants containing the L83V amino acid mutation are more frequently detected in cervical cancer than the corresponding prototype HPV 16 as evidenced by independent epidemiological data. We set out to analyze in a cell culture study how these variants in the absence of E7 perform during vital steps of carcinogenesis and assessed their ability to immortalize and transform primary human foreskin keratinocytes (PHFKs). Their migration ability, a hallmark for invasiveness and/or metastasis, was also investigated. Immortalization capability was based on population doublings, number of passages, surpassing Mortality Stages 1 and 2, telomerase reverse transcriptase expression and the ability to overcome G1 arrest via p53 degradation. Transformation and migration efficiency were analyzed by a combination of functional cell-based assays. For the first time, we observed that all E6 proteins alone were sufficient to immortalize PHFKs, with the AAE6 variant being the most proficient. The AAE6 variant protein alone also pushed PHFKs through transformation and significantly increased their migration ability over that of the PHFKs expressing L83V and E6 prototype proteins. Our findings are in line with epidemiological data that the AAE6 variant confers an increased risk over the prototype for cervical cancer as evidenced by a superior immortalization, transformation and metastasis potential

THE EFFECTS OF EXERCISE ON COGNITIVE FUNCTION, AND BALANCE IN HEALTHY INDIVIDUALS - A PILOT STUDY

Concussion is defined as an injury to the brain caused by forces applied to the head. Approximately 10-20% of concussed individuals develop post-concussion syndrome (PCS), characterized by persistent symptoms beyond 10 days including balance and cognitive deficits. The purpose of this pilot study was to explore the effect of a four-week exercise program on physiological, cognitive, and balance variables in a sample of healthy, physically active individuals to gather preliminary information to apply to a future PCS population. Statistically significant changes in reaction time and balance measures were observed. No changes in heart rate, blood pressure, memory, or visual motor speed were observed. Balance improvements in this healthy sample suggest that future exploration of a similar exercise program in those experiencing PCS may provide valuable information

STATIC BALANCE IN INDIVIDUALS WITH POST-CONCUSSION SYNDROME

A concussion is an injury to the brain caused by linear and/or rotational forces applied to the head. The majority of concussions typically resolve spontaneously within a period of 7-10 days. In 10-20% of individuals, however, the symptoms persist beyond 10 days and are termed post-concussion syndrome (PCS). The purpose of this pilot study was to evaluate the effects of a supervised and structured four-week aerobic and balance exercise program on static balance measures in a sample of individuals with PCS. Statistically significant changes in velocity and length of centre of pressure during balancing tasks were observed in response to the exercise program. These improvements in balance and symptoms suggest that further research into the benefits of exercise based treatment for improving balance deficits associated with PCS is warranted

Toll-Like Receptor Transcriptome in the HPV-Positive Cervical Cancer Microenvironment

The human papillomavirus (HPV) directly infects cervical keratinocytes and interferes with TLR signalling. To shed light on the effect of HPV on upstream receptors, we evaluated TLRs 1–9 gene expression in HPV-negative normal and HPV-positive pre-malignant and malignant ex vivo cervical tissue. Quantitative real-time polymerase chain reaction was performed separately for epithelial and stromal tissue compartments. Differences in gene expression were analyzed by the Jonckheere-Terpstra trend test or the Student's t-test for pairwise comparison. Laser capture microdissection revealed an increase in TLR3 and a decrease in TLR1 mRNA levels in dysplastic and carcinoma epithelium, respectively. In the stroma, a trend of increasing TLR 1, 2, 5, 6, and 9 mRNA levels with disease severity was found. These findings implicate the involvement of TLR3 and TLR1 in early and late cervical carcinogenesis, respectively, suggesting that stromal upregulation of TLRs may play a role in cervical disease progression

Enhanced Longevity by Ibuprofen, Conserved in Multiple Species, Occurs in Yeast through Inhibition of Tryptophan Import

The common non-steroidal anti-inflammatory drug ibuprofen has been associated with a reduced risk of some age-related pathologies. However, a general pro-longevity role for ibuprofen and its mechanistic basis remains unclear. Here we show that ibuprofen increased the lifespan of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster, indicative of conserved eukaryotic longevity effects. Studies in yeast indicate that ibuprofen destabilizes the Tat2p permease and inhibits tryptophan uptake. Loss of Tat2p increased replicative lifespan (RLS), but ibuprofen did not increase RLS when Tat2p was stabilized or in an already long-lived strain background impaired for aromatic amino acid uptake. Concomitant with lifespan extension, ibuprofen moderately reduced cell size at birth, leading to a delay in the G1 phase of the cell cycle. Similar changes in cell cycle progression were evident in a large dataset of replicatively long-lived yeast deletion strains. These results point to fundamental cell cycle signatures linked with longevity, implicate aromatic amino acid import in aging and identify a largely safe drug that extends lifespan across different kingdoms of life.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

The human papillomavirus 16 European-T350G E6 variant can immortalize but not transform keratinocytes in the absence of E7

AbstractHuman papillomavirus type 16 is commonly implicated in HPV-related cancers. However, only a small number of infected individuals progress to this stage. Epidemiological evidence demonstrated that oncogenic risk is population-specific and variations within the viral oncogene, E6, have been suggested to play a role in these findings. Of focus in this study is the European-T350G variant, which is characterized by an L>V amino acid substitution at residue 83 of the prototype E6 protein. To elucidate the functional effects of this polymorphism, we followed keratinocytes transduced with E-T350G E6 for over 60 passages and compared them to keratinocytes transduced, in parallel, with prototype or Asian-American (Q14H/L83V/H78Y) E6. We found that although E-T350G E6 immortalized transduced keratinocytes in the absence of E7, these cells were not fully transformed. We also found that E-T350G down-regulated E-cadherin compared to the other variants, providing a possible link between its population-based oncogenicity and host genetic variations

Leucine Potentiates Glucose-mediated 18F-FDG Uptake in Brown Adipose Tissue via β-Adrenergic Activation

Significant depots of brown adipose tissue (BAT) have been identified in many adult humans through positron emission tomography (PET), with the amount of BAT being inversely correlated with obesity. As dietary activation of BAT has implications for whole body glucose metabolism, leucine was used in the present study to determine its ability to promote BAT activation resulting in increased glucose uptake. In order to assess this, 2-deoxy-2-(fluorine-18)fluoro-d-glucose (18F-FDG) uptake was measured in C57BL/6 mice using microPET after treatment with leucine, glucose, or both in interscapular BAT (IBAT). Pretreatment with propranolol (PRP) was used to determine the role of β-adrenergic activation in glucose and leucine-mediated 18F-FDG uptake. Analysis of maximum standardized uptake values (SUVMAX) determined that glucose administration increased 18F-FDG uptake in IBAT by 25.3%. While leucine did not promote 18F-FDG uptake alone, it did potentiate glucose-mediated 18F-FDG uptake, increasing 18F-FDG uptake in IBAT by 22.5%, compared to glucose alone. Pretreatment with PRP prevented the increase in IBAT 18F-FDG uptake following the combination of glucose and leucine administration. These data suggest that leucine is effective in promoting BAT 18F-FDG uptake through β-adrenergic activation in combination with glucose

Sense and antisense RNA are not toxic in <i>Drosophila </i>models of <i>C9orf72</i>-associated ALS/FTD

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Neurodegeneration may occur via transcription of the repeats into inherently toxic repetitive sense and antisense RNA species, or via repeat-associated non-ATG initiated translation (RANT) of sense and antisense RNA into toxic dipeptide repeat proteins. We have previously demonstrated that regular interspersion of repeat RNA with stop codons prevents RANT (RNA-only models), allowing us to study the role of repeat RNA in isolation. Here we have created novel RNA-only Drosophila models, including the first models of antisense repeat toxicity, and flies expressing extremely large repeats, within the range observed in patients. We generated flies expressing ~ 100 repeat sense or antisense RNA either as part of a processed polyadenylated transcript or intronic sequence. We additionally created Drosophila expressing > 1000 RNA-only repeats in the sense direction. When expressed in adult Drosophila neurons polyadenylated repeat RNA is largely cytoplasmic in localisation, whilst intronic repeat RNA forms intranuclear RNA foci, as does > 1000 repeat RNA, thus allowing us to investigate both nuclear and cytoplasmic RNA toxicity. We confirmed that these RNA foci are capable of sequestering endogenous Drosophila RNA-binding proteins, and that the production of dipeptide proteins (poly-glycine-proline, and poly-glycine-arginine) is suppressed in our models. We find that neither cytoplasmic nor nuclear sense or antisense RNA are toxic when expressed in adult Drosophila neurons, suggesting they have a limited role in disease pathogenesis

Lactate production from HeLa cells exposed to 2-deoxy-D-glucose (2DG) or non-radioactive 2-deoxy-2-fluoro-D-glucose (¹⁹FDG) under either normoxic or hypoxic (1% O₂) conditions.

<p>HeLa cells were treated with different doses of 2DG or ¹⁹FDG for 24 hours under either normoxia (A) or hypoxia (B), and lactate production was measured from the cell culture media. Lactate production was normalized to the amount of protein from the corresponding cells. n = 3, * denotes significance from 2DG within the same glucose analog concentration, p≤0.05. All doses of both analogs significantly decreased lactate production of cells compared to untreated cells.</p

Glucose uptake inhibition in MDA-MB-231 tumours <i>in vivo</i>.

<p>A solution of five million MDA-MB-231 cells in serum free media was injected into the right leg/flank area of athymic nude mice. Tumours were allowed to grow for 3–4 weeks before mice were imaged. Prior to imaging, mice were fasted 5 hours. Once tumours reached an SUVmax of at least 1 (deemed “week 1”), mice were placed into either the control group or the non-radioactive 2-deoxy-2-fluoro-D-glucose (¹⁹FDG) group. The ¹⁹FDG group received 220mg/kg ¹⁹FDG 2 hours prior to ¹⁸F-FDG. Intraperitoneal saline injections were used as an injection control in the control group mice. 20μCi ¹⁸F-FDG was injected into the intraperitoneal cavity of all mice 1 hour prior to imaging with the microPET. The second round of imaging took place one week after the first. Panel A shows SUVmax results from week 2 as a percentage of the week 1 imaging results. n = 2–3, * denotes significance from control mice, p≤0.05. Panel B shows representative reconstructed microPET images of mice following week one and week two of treatment. The white arrow highlights the tumour location.</p