Increased DNA methyltransferase activity and DNA methylation following epidermal growth factor stimulation in ovarian cancer cells

Ovarian cancer progression is correlated with accumulation of aberrant CpG island methylation. In ovarian cancer, ascites fluid contains numerous Epidermal-Growth-Factor-Receptor (EGFR) activators, which could result in a tumor microenvironment of constant EGFR activation. Signaling pathways downstream of EGFR, such as Ras, regulate DNA methylation. We hypothesized that chronic EGFR activation could alter DNA methylation. We found that EGFR activation increased DNA methyltransferase (DNMT) activity acutely, as well as after long-term EGF treatment or expression of a mutationally activated EGFR. Furthermore, this increase in DNMT activity was dependent on EGFR catalytic activity and resulted in increased global DNA methylation. Additionally, treatment with the DNMT inhibitor/hypomethylating agent 5-Aza-2′-deoxycytidine (AZA) inhibited the EGF induced increase of both DNMT activity and global methylation. These data support a role for EGFR in the process of accumulated DNA methylation during ovarian cancer progression and suggest that epigenetic therapy may be beneficial for the treatment of ovarian cancer.Key words: ovarian cancer, DNA methylation, epidermal growth factor receptor, DNA methyltransferase, epigenetics, E-cadherin     

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth^1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect^1,2, mammalian^3-5, and human cells6 including primary human T cells^7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency^9,10.Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer¹¹. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved¹². We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene⁷. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes¹³. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model¹⁴. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized¹⁵.Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens¹⁴, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed⁷. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)¹⁵. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.     

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults ¹. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via β adrenergic receptor ^{2, 3}. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters ^4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance ^6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT ^9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of ¹⁸F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of β-adrenoceptor antagonist propranolol ^{14, 15}, or the enhanced BAT activation by β3 agonist BRL37344 ¹⁶. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.     

Comparison of an in-helmet temperature monitor system to rectal temperature during exercise

Body temperature monitoring is crucial in helping to decrease the amount and severity of heat illnesses; however, a practical method of monitoring temperature is lacking. In response to the lack of a practical method of monitoring the temperature of athletes, Hothead Technologies developed a device (HOT), which continuously monitors an athlete's fluctuations in body temperature. HOT measures forehead temperature inside helmets. The purpose of this study was to compare HOT against rectal temperature (Trec). Male volunteers (n = 29, age = 23.5 ± 4.5 years, weight = 83.8 ± 10.4 kg, height = 180.1 ± 5.8 cm, body fat = 12.3 ± 4.5%) exercised on a treadmill at an intensity of 60-75% heart rate reserve (HRR) (wet bulb globe temperature [WBGT] = 28.7° C) until Trec reached 38.7° C. The correlation between Trec and HOT was 0.801 (R = 0.64, standard error of the estimate (SEE) = 0.25, p = 0.00). One reason for this relatively high correlation is the microclimate that HOT is monitoring. HOT is not affected by the external climate greatly because of its location in the helmet. Therefore, factors such as evaporation do not alter HOT temperature to a great degree. HOT was compared with Trec in a controlled setting, and the exercise used in this study was moderate aerobic exercise, very unlike that used in football. In a controlled laboratory setting, the relationship between HOT and Trec showed favorable correlations. However, in applied settings, helmets are repeatedly removed and replaced forcing HOT to equilibrate to forehead temperature every time the helmet is replaced. Therefore, future studies are needed to mimic how HOT will be used in field situations.     

A preliminary examination of the diet and feeding of sea sweep Scorpis aequipinnis (Pisces: Scorpididae) from coastal waters of south-western Victoria, Australia

The diet and feeding of sea sweep (Scorpis aequipinnis) was investigated from 230 specimens collected from the south-western Victorian coastline between January and July, 2002. Stomach content analysis indicated that S. aequipinnis are browsing omnivores (55.6% algae, 25.4% animal, by dry weight), with rhodophytes found to be the most important component of their diet (93.5% frequency of occurrence; 42.6% dry weight; 45.5% prey-specific abundance). However, the relative proportions of the major dietary components differed significantly between size classes. The dietary composition of small individuals ( < 150 mm total length) was significantly different to larger individuals, primarily due to a higher degree of carnivory exhibited by the smaller fish. S. aequipinnis were found to be highly flexible feeders exploiting both benthic and pelagic food resources, characterised by irregular periods of selective carnivorous feeding.     

Short-term UV-B radiation and ozone exposure effects on aromatic secondary metabolite accumulation and shoot growth of flavonoid-deficient Arabidopsis mutants

The presence of UV-absorptive substances in the epidermal cells of leaves is thought to protect mesophyll tissues from the harmful effects of UV-B radiation. We examined the influence of short-term UV-B exposures on UV-absorptive (330 nm) sinapates and flavonols, and on shoot growth of the Arabidopsis wild type ecotype Landsberg erecta and two mutants. 114 deficient in chalcone synthase, and 115 , deficient in chalcone/flavonone isomerase. Sequential ozone exposures were used to determine the effects of oxidative stress The levels of sinapates and flavonols on a leaf fresh weight basis increased substantially in the wild type and sinapates increased in the 114 mutant in vegetative vegetative/reproductive transitional and reproductive stage plants in response to short-term (48h) UV-B radiation. When UV-B was discontinued the levels generally decreased lo pre-exposure levels after 48 h in vegetative/reproductive but not in reproductive plants. Exposure to ozone before or alter UV-B treatment did not consistently affect the levels of these UV-absorptive compounds. Dry matter accumulation was less affected by UV-B at the vegetative and reproductive stages than at the vegetative/reproductive stage. At the vegetative/reproductive stage, shoot growth of all 3 genotypes was retarded by UV-B. Growth was not retarded by short-term ozone exposure alone but when exposure to ozone followed UV-B exposure, growth was reduced in all genotypes. Leaf cupping appeared on 115 plants exposed to UV-B.     

Nervous system modification by transplants and gene transfer

New possibilities to modify function and direct repair in the central nervous system (CNS) have been established by the merger of gene transfer technology with neural transplantation. Rapid advances in viral-mediated DNA-delivery procedures permit the study of novel gene expression in neurons and glial cells. Foreign genes, transferred by a virus vector, can be used to generate new cell lines, identify transplanted cells, and express growth factors or enzymes for neurotransmitter synthesis. In addition to CNS cell types, non-neural cells are also being studied with transgene technology in the nervous system. Functional effects have been obtained with grafts of genetically modified cells in animal models of several nervous system disorders, and the recent results set the stage for potential application of these techniques to human CNS gene therapy.     

Reflexive governance in biobanking: on the value of policy led approaches and the need to recognise the limits of law

Although a few jurisdictions around the world have legislated in response to the phenomenon of biobanking, the far more common response has been policy led with funders and other stakeholders initiating multi-level policy initiatives to guide biobanking practice. An example of this is UK Biobank which has developed and operates according to an Ethics and Governance Framework. Such an instrument has no basis in law and yet it has played a crucial role in the set up and ongoing management of the resource. It will continue to do so, as related policies emerge, such as access and intellectual property policies. Numerous biobanking initiatives have similar high-level policy documents that guide decisions and practice. These are often framed as a commitment to participants, researchers and society more broadly and invoke notions such as the public good and the public interest. As such, they serve as a benchmark against which to measure a biobank’s performance. Moreover, policies become an important means by which biobankers are held accountable. This article critically analyses this policy-driven phenomenon asking how effectively policy—often as an alternative to law—serves to police and to promote biobanking. It argues that a policy of reflexive governance—defined and developed herein—can best meet the challenges faced by many biobanks and without the need for recourse to law.     

Morphometrics and pattern of growth in wild sifakas (Propithecus edwardsi) at ranomafana national park,madagascar

We summarize morphometric data collected over a period of 22 years from a natural population of rainforest sifakas (Propithecus edwardsi) at Ranomafana National Park, Madagascar, and we use those data to document patterns of growth and development. Individually identified, known‐age sifakas were successfully captured, measured, and released. We found that body segment lengths increased faster during growth than did body mass, with individuals attaining adult lengths earlier than adult mass. Females can begin reproducing before they are fully grown, but this may not be common. With the exception of hand length, we found no significant sex difference in any adult metric including body mass, chest, and limb circumferences, body segment lengths, and canine tooth height; however, body masses of individual females fluctuated more, independently of pregnancy, than did those of males. We found considerable interannual fluctuation in body mass with single individuals differing more within the same season in different years than from season to season in the same year. Such body mass fluctuation may be a consequence of eastern Madagascar's variable and unpredictable environment in which rainfall during any selected month varies from year to year. Am. J. Primatol. 73:155–172, 2011. © 2010 Wiley‐Liss, Inc.