49 research outputs found
Chromosomal localization of a proinsulin transgene inserted with a transposon-based vector into Japanese quail, Coturnix coturnix
The overall goals of this research were to develop a reproducible method of detecting stable DNA insertion into Japanese quail and provide a method for gene location on avian chromosomes. This research resulted in the development of a different method of obtaining chromosome spreads in Japanese quail, the establishment of primed in situ hybridization as a method for the chromosomal gene detection in birds, development of Teflon-coated coverslip slides to facilitate laser microdissection of 0.5 ƒÝm samples, and chromosomal identification of proinsulin transgene insertions by laser microdissection and nucleotide sequence from G2 Japanese quail. The 28S rDNA was found on a macrochromosome and a microchromosome pair by primed in situ hybridization, fluorescent in situ hybridization, and silver staining. Teflon-coated coverslip slides were created to facilitate laser microdissection of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G2 Japanese quail produced in Dr. Richard Cooper¡¦s laboratory were identified by laser microdissection and found to have 2-5 chromosomal insertions of the proinsulin transgene
The mammalian centrosome and its functional significance
Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome’s functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome’s role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome’s significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease
The Formation of the First Stars in the Universe
In this review, I survey our current understanding of how the very first
stars in the universe formed, with a focus on three main areas of interest: the
formation of the first protogalaxies and the cooling of gas within them, the
nature and extent of fragmentation within the cool gas, and the physics -- in
particular the interplay between protostellar accretion and protostellar
feedback -- that serves to determine the final stellar mass.
In each of these areas, I have attempted to show how our thinking has
developed over recent years, aided in large part by the increasing ease with
which we can now perform detailed numerical simulations of primordial star
formation. I have also tried to indicate the areas where our understanding
remains incomplete, and to identify some of the most important unsolved
problems.Comment: 74 pages, 4 figures. Accepted for publication in Space Science
Review
On the issue of transparency and reproducibility in nanomedicine.
Following our call to join in the discussion over the suitability of implementing a reporting checklist for bio-nano papers, the community responds
Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial
Background
Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear.
Methods
RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047.
Findings
Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths.
Interpretation
Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population
Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial
Background
Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain.
Methods
RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and
ClinicalTrials.gov
,
NCT00541047
.
Findings
Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths.
Interpretation
Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy.
Funding
Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society
Chromosomal localization of a proinsulin transgene in Japanese quail by laser pressure catapulting
Transgenic avian bioreactors produce therapeutic recombinant proteins in egg white. To date, however, methods for transgenic modification of the avian genome or determining transgenic status of individual birds are scarce. The dual, but interrelated, goals of this research were to: (1) develop a method of detecting stable DNA insertion into Japanese quail; and (2) provide a method for gene location on avian chromosomes. We created Teflon-coated coverslip slides to facilitate laser pressure catapulting of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G2 Japanese quail, containing germline incorporation of proinsulin, were identified by isolation of chromosomes using laser microdissection and laser pressure catapulting. Subsequent amplification of each chromosome identified 2-5 chromosomes with the proinsulin transgene inserted. Nucleotide sequencing of each chromosomal insertion was identical to the proinsulin portion of the original vector. By applying laser pressure catapulting and PCR of individual chromosomes, we were able to determine that the transgene correctly inserted into avian chromosomes and that the majority of the insertions occurred within microchromosomes. Because many potential therapeutic transgenes have similar or nearly identical nucleotide sequence to the host\u27s native gene, laser microdissection and subsequent analysis may be required for detailed documentation of transgene expression before proceeding with transgenic protein production. © Springer Science+Business Media B.V. 2006
Chloroquine-Mediated Cell Death in Metastatic Pancreatic Adenocarcinoma Through Inhibition of Autophagy
Context Cells in the interior of pancreatic tumors are believed to undergo continual autophagy to maintain homeostasis during unregulated growth in hypoxia caused by impaired vascularity. We hypothesize that treating metastatic cells with chloroquine, an inhibitor of autophagy, in hypoxia will decrease cell viability and induce cell death. Design MiaPaCa2 (non-metastatic) and S2VP10 (metastatic) cell lines were treated with 25 and 50 µM chloroquine for 24 and 48 hours in normoxia and hypoxia (5-10% O2). Viability was measured using ATPliteTM. After treatment, the cell stress was analyzed, and protein was lysed and quantified using the Bradford assay. Autophagy-associated protein levels were determined by Western blot. Results S2VP10 cells treated for 48 hours with 50 μM chloroquine in hypoxia had 24% viability compared to normoxia control, with loss of 10% viability caused by low O2 alone. MiaPaCa2 cells under these conditions had 60% viability compared to normoxia control, with loss of 25% viability caused by low O2 alone. Analysis of cell stress pathways and dosimetry of Western blot data suggest that chloroquine inhibits the autophagy pathway in the metastatic S2VP10 cells. Conclusion Autophagy blockage with chloroquine or similar-acting drugs may serve as a viable therapy for highly metastatic pancreatic cancers.Image: MiaPaCa2 cells treated with 25 μM chloroquine for 24 hours (Figure 1e
Small Molecule Optoacoustic Contrast Agents: An Unexplored Avenue for Enhancing In Vivo Imaging
Almost every variety of medical imaging technique relies heavily on exogenous contrast agents to generate high-resolution images of biological structures. Organic small molecule contrast agents, in particular, are well suited for biomedical imaging applications due to their favorable biocompatibility and amenability to structural modification. PET/SPECT, MRI, and fluorescence imaging all have a large host of small molecule contrast agents developed for them, and there exists an academic understanding of how these compounds can be developed. Optoacoustic imaging is a relatively newer imaging technique and, as such, lacks well-established small molecule contrast agents; many of the contrast agents used are the same ones which have found use in fluorescence imaging applications. Many commonly-used fluorescent dyes have found successful application in optoacoustic imaging, but others generate no detectable signal. Moreover, the structural features that either enable a molecule to generate a detectable optoacoustic signal or prevent it from doing so are poorly understood, so design of new contrast agents lacks direction. This review aims to compile the small molecule optoacoustic contrast agents that have been successfully employed in the literature to bridge the information gap between molecular design and optoacoustic signal generation. The information contained within will help to provide direction for the future synthesis of optoacoustic contrast agents