Establishment of a doxycycline-regulated cell line with inducible, doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells

p53 is important in the development of hepatocellular carcinoma (HCC) and in therapeutic approaches, but the mechanism whereby it inhibits HCC growth is still unclear. The aim of the present study was to establish a HCC cell system in which p53 levels can be regulated. Full-length wild-type p53 cDNA obtained by PCR was cloned into a retroviral response vector controlled by the tetracycline responsive element (RevTRE-p53). The regulatory vectors RevTet-Off and RevTRE-p53 were transfected into a packaging cell line, PT67. Hep3B cells in which the p53 gene was deleted were infected with RevTet-Off viral particles from the PT67. Three G418-resistant cell clones with high luciferase expression and low background were infected with RevTRE-p53. By screening dozens of RevTRE-p53-infected clones with hygromycin we identified the one with the highest expression of p53 and the lowest background after doxycycline treatment. The results showed that p53 expression in this cell clone could be simply turned on or off by removing or adding doxycycline. Furthermore, it was found that the level of p53 protein was negatively and sensitively related to the doxycycline concentration. In conclusion, we have established a HCC cell line in which p53 expression can be switched on or off and regulated in a dose- and time-dependent manner

Association of CYP2C19*17 Allele and Choice of P2Y12 Inhibitor on Cardiovascular Outcomes Following Percutaneous Coronary Intervention

Introduction: The CYP2C19*17 allele variant is a gain-of-function polymorphism which increases levels of the active metabolite of clopidogrel. Objective: *17 is associated with increased bleeding risk during clopidogrel therapy, but it is unclear whether alternative P2Y12 inhibitors, prasugrel and ticagrelor, produce better clinical outcomes in patients undergoing percutaneous coronary intervention (PCI). Methods: A single-center observational study was conducted in 928 PCI patients who received CYP2C19 testing and dual anti-platelet therapy (DAPT). Risk of major adverse cardiovascular or cerebrovascular events (MACCE) and clinically significant bleeding over 12 months were compared across genotype and DAPT groups by proportional hazards regression. Results: 584 patients were treated with clopidogrel while 344 patients had alternative therapy. In the clopidogrel group, 173 patients were hetero- or homozygous for *17 and 91 patients were hetero- or homozygous for a loss of function allele (LOF; *2 or *3). Patients treated with clopidogrel were older, more commonly female, and more likely to have hypertension, diabetes, and an acute coronary syndrome (ACS) than patients on alternative therapy. There were no differences in MACCE or clinically significant bleeding events in *17 patients treated with clopidogrel compared to alternative therapy in either the total population (p=0.54) or in ACS patients (p=0.98). Patients with LOF alleles were 3.4 times more likely in the total population (p \u3c0.0001) and 6.7 times more likely among ACS patients (p \u3c0.0001) to have MACCE if prescribed clopidogrel compared with alternative therapy. Discussion: *17 patients had equivalent clinical outcomes when treated with clopidogrel or alternative P2Y12 inhibitors

Indiana Emergency Medical Services Needs Assessment: Workforce and Training Issues

A mail survey was conducted to assess issues related to the EMS workforce and EMS workforce training in Indiana. Responses were received from approximately 45% of individuals and EMS organization contacted. Major findings are: Evidence exists of a shortage of EMS personnel in Indiana due to high vacancy rates. This is particularly acute in volunteer organizations, which report the greatest difficulty in recruiting EMS personnel. Paid EMS personnel work on average significantly more than a standard 40 hour week, and high percentages of EMS personnel add a second job. Because excessive working hours have been linked to low quality healthcare, this may indicate potential problems with the quality of EMS care. Differences in training needs and training received in areas related to cardiac care indicate potential disparities in the quality of care for heart attacks between rural and urban areas. Contrary to the Indiana requirements for recertification, it appears that a significant proportion of Indiana EMS personnel and organizations are not using audits as a tool for ensuring quality and developing skills. A wide variety of training needs have been identified. While some clinical skill areas (basic and advanced life support) are needed, many of these areas are in important non-clinical areas, such as automobile extrication, foreign languages, safety, and incident management. The training environment, both in terms of initial and continuing training, is so varied that it cannot realistically be considered a “system.” In the abstract, EMS personnel prefer “hands-on” type training, but when presented with specific scenarios they indicate a greater affinity for locally-delivered mechanisms such as classroom courses at a local school or computer-based training. This probably reflects the tradeoff between time and utility, and is consistent with findings that time is the largest barrier to continuing education and the high percentage EMS personnel who hold second jobs. Self-assessment of preparedness levels by EMS personnel reveals that the EMS system in rural areas is likely to be less prepared and capable than in urban areas. Volunteer organizations rate themselves as less capable than paid organizations, although this is largely not reflected in self-assessments by volunteer EMS personnel

Should The US Media Have A Duty To Cover All International Tragedies Even If Such Reporting Cannot Be Measured In Their Ratings and, Thereby, Their Respective Financial Status?

Hurricane Katrina was one of the most devastating natural disasters to occur in United States history. Within months, both the domestic and international news media has moved on to more lucrative stories that inspire higher ratings. The international media, has been criticized for failing to provide balanced and ethical coverage of international disasters, and has focused only on reporting those news items that can be measured in their ratings and has a positive impact on the financial bottom line

Investigation of Superconducting Gap Structure in TbFeAsO0.9_{0.9}F0.1_{0.1} using Point Contact Andreev Reflection

Bulk samples of TbFeAsO$_{0.9}$F$_{0.1}$ (T$_{c}$(on) = 50K) were measured by point contact Andreev reflection spectroscopy. The spectra show unambiguous evidence for multiple gap-like features plus the presence of high bias shoulders. By measuring the spectra as a function of temperature with both gold and superconducting niobium tips, we establish that the gap-like features are associated with superconducting order parameter in this material. We discuss whether the well defined zero bias conductance peak that we observe infrequently is associated with a nodal superconducting order parameter.Comment: 11 pages, 5 figures, published versio

Multicolour correlative imaging using phosphor probes

Correlative light and electron microscopy exploits the advantages of optical methods, such as multicolour probes and their use in hydrated live biological samples, to locate functional units, which are then correlated with structural details that can be revealed by the superior resolution of electron microscopes. One difficulty is locating the area imaged by the electron beam in the much larger optical field of view. Multifunctional probes that can be imaged in both modalities and thus register the two images are required. Phosphor materials give cathodoluminescence (CL) optical emissions under electron excitation. Lanthanum phosphate containing thulium or terbium or europium emits narrow bands in the blue, green and red regions of the CL spectrum; they may be synthesised with very uniform-sized crystals in the 10- to 50-nm range. Such crystals can be imaged by CL in the electron microscope, at resolutions limited by the particle size, and with colour discrimination to identify different probes. These materials also give emissions in the optical microscope, by multiphoton excitation. They have been deposited on the surface of glioblastoma cells and imaged by CL. Gadolinium oxysulphide doped with terbium emits green photons by either ultraviolet or electron excitation. Sixty-nanometre crystals of this phosphor have been imaged in the atmospheric scanning electron microscope (JEOL ClairScope). This probe and microscope combination allow correlative imaging in hydrated samples. Phosphor probes should prove to be very useful in correlative light and electron microscopy, as fiducial markers to assist in image registration, and in high/super resolution imaging studies

Predicting treatment outcome for incompetent defendants

This studv examined the ~rediction of outcome in a sample of defendants hospitalized i o r treatment of incompetency. Defendants' demog;aphic characteristics and scores on 18 scales of the Computer-Assisted Determination of Competency to Proceed instrument (CADCOMP) were used to predict competency restoration and length of stay (LOS). During the period of study, almost 90 percent of the defendants were restored to competency after a mean stay of over 280 days. Demographic characteristics were unrelated to outcome. Several CADCOMP scales, including two scales measuring psycholegal ability and one measuring psychopathology, were correlated with both outcome criteria. Discriminant analysis using the CADCOMP scales accurately classified 76.7 percent of the defendants into short and long stay groups. Although promising, the findings are nevertheless consistent with prior research in suggesting that examiners should exercise caution in providing feedback to the courts concerning competency restoration and the period of time needed for treatment

Mammalian Stem Cells Reprogramming in Response to Terahertz Radiation

We report that extended exposure to broad-spectrum terahertz radiation results in specific changes in cellular functions that are closely related to DNA-directed gene transcription. Our gene chip survey of gene expression shows that whereas 89% of the protein coding genes in mouse stem cells do not respond to the applied terahertz radiation, certain genes are activated, while other are repressed. RT-PCR experiments with selected gene probes corresponding to transcripts in the three groups of genes detail the gene specific effect. The response was not only gene specific but also irradiation conditions dependent. Our findings suggest that the applied terahertz irradiation accelerates cell differentiation toward adipose phenotype by activating the transcription factor peroxisome proliferator-activated receptor gamma (PPARG). Finally, our molecular dynamics computer simulations indicate that the local breathing dynamics of the PPARG promoter DNA coincides with the gene specific response to the THz radiation. We propose that THz radiation is a potential tool for cellular reprogramming

Search for Exotic Strange Quark Matter in High Energy Nuclear Reactions

We report on a search for metastable positively and negatively charged states of strange quark matter in Au+Pb reactions at 11.6 A GeV/c in experiment E864. We have sampled approximately six billion 10% most central Au+Pb interactions and have observed no strangelet states (baryon number A < 100 droplets of strange quark matter). We thus set upper limits on the production of these exotic states at the level of 1-6 x 10^{-8} per central collision. These limits are the best and most model independent for this colliding system. We discuss the implications of our results on strangelet production mechanisms, and also on the stability question of strange quark matter.Comment: 21 pages, 9 figures, to be published in Nuclear Physics A (Carl Dover memorial edition