DNA dosimeter measurements of beam profile using a novel simultaneous processing technique.

A DNA dosimeter (DNAd) was previously developed that uses double-strand breaks (DSB) to measure dose. This dosimeter has been tested to measure dose in scenarios where transient-charged particle equilibrium (TCPE) has been established. The probability of double strand break (PDSBo), which is the ratio of broken double-stranded DNA (dsDNA) to the initial unbroken dsDNA in the dosimeter, was used to quantify DSBs and related to dose. The goal of this work is to produce a new technique to process and analyze the DNAd and quantify DNA-DSBs. This technique included simultaneously processing multiple DNAds and also establishing a new form to the probability of double strand break (PDSBn), which was then used to test the DNAd in a non-TCPE condition by taking beam penumbra measurements. The technique utilized a 384-well plate, and the measurements were made at the edge of a 10 × 10 cm field and compared to film measurements. During these penumbra measurements, while observing the positional differences in the higher gradient region at 4.1 and 4.55 cm from the center of the radiation field, the distance to agreement of PDSBo to film were 0.38 cm and 0.26 cm while the distance to agreement of PDSBn to film were 0.11 cm and 0.06 cm, respectively. Finally, the developed new separation technique reduced the time needed for the analysis of 25 samples from 200 min to 30 min.

Optimizing the response, precision, and cost of a DNA double-strand break dosimeter.

We developed a dosimeter that measures biological damage following delivery of therapeutic beams in the form of double-strand breaks (DSBs) to DNA. The dosimeter contains DNA strands that are labeled on one end with biotin and on the other with fluorescein and attached to magnetic microbeads. Following irradiation, a magnet is used to separate broken from unbroken DNA strands. Then, fluorescence is utilized to measure the relative amount of broken DNA and determine the probability for DSB. The long-term goal for this research is to evaluate whether this type of biologically based dosimeter holds any advantages over the conventional techniques. The purpose of this work was to optimize the dosimeter fabrication and usage to enable higher precision for the long-term research goal. More specifically, the goal was to optimize the DNA dosimeter using three metrics: the response, precision, and cost per dosimeter. Six aspects of the dosimeter fabrication and usage were varied and evaluated for their effect on the metrics: (1) the type of magnetic microbeads, (2) the microbead to DNA mass ratio at attachment, (3) the type of suspension buffer used during irradiation, (4) the concentration of the DNA dosimeter during irradiation, (5) the time waited between fabrication and irradiation of the dosimeter, and (6) the time waited between irradiation and read out of the response. In brief, the best results were achieved with the dosimeter when attaching 4.2 µg of DNA with 1 mg of MyOne T1 microbeads and by suspending the microbead-connected DNA strands with 200 µl of phosphate-buffered saline for irradiation. Also, better results were achieved when waiting a day after fabrication before irradiating the dosimeter and also waiting an hour after irradiation to measure the response. This manuscript is meant to serve as guide for others who would like to replicate this DNA dose measurement technique.

Cartilage oligomeric matrix protein-angiopoientin-1 enhances angiogenesis of isolated islet and maintains normoglycemia following transplantation.

Islet transplantation (ITx) has potential as a therapy for patients with type 1 diabetes. For successful engraftment and insulin independence, the transplanted islets must establish an adequate, stable blood supply. Angiopoientin-1 (Ang1) is a specific growth factor that induces vascularization via the Tie2 or Tie1 receptor. In this study, we used an in vitro angiogenesis assay to evaluate islet function following transplantation and the effect of the Ang1 variant cartilage oligomeric matrix protein (COMP) Ang1 on isolated islets. The enhanced function of islets transduced with COMP-Ang1 was also confirmed in a streptozotocin (STZ)-induced diabetic mice model. In a three-dimensional collagen-based culture system, the transduction of COMP-Ang1 into islets significantly increased angiogenesis compared with the bacterial-ß-galactosidase (LacZ)-transduced controls and an intact, nontransduced islet negative control group. COMP-Ang1 transduced islets also attenuated hyperglycemia in syngeneic diabetic C57BL/6 mice and enhanced glucose tolerance by areas under the curves of intraperitoneal glucose tolerance tests. These findings demonstrated the capacity of COMP-Ang1 to promote revascularization in cultured islets, which may contribute to successful transplantation in vivo.

cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans.

A high frequency of apoptosis is a conserved hallmark of oocyte development. In C. elegans, about half of all developing oocytes are normally killed by a physiological germline-specific apoptosis pathway, apparently so that they donate cytoplasm to the survivors. We have investigated the functions of CGH-1, the C. elegans ortholog of the predicted RNA helicase ste13/ME31B/RCK/p54, which is germline-associated in metazoans and required for sexual reproduction in yeast. We show that CGH-1 is expressed specifically in the germline and early embryo, and is localized to P granules and other possible mRNA-protein particles. cgh-1 is required for oocyte and sperm function. It is also needed to prevent the physiological germline apoptosis mechanism killing essentially all developing oocytes, making lack of cgh-1 function the first stimulus identified that can trigger this mechanism. We conclude that cgh-1 and its orthologs may perform conserved functions during gametogenesis, that in C. elegans certain aspects of oocyte development are monitored by the physiological germline apoptosis pathway, and that similar surveillance mechanisms may contribute to germline apoptosis in other species.

Transcriptional repression by the Caenorhabditis elegans germ-line protein PIE-1.

In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.

Nucleosome positioning by the winged helix transcription factor HNF3.

Nucleosome positioning at genetic regulatory sequences is not well understood. The transcriptional enhancer of the mouse serum albumin gene is active in liver, where regulatory factors occupy their target sites on three nucleosome-like particles designated N1, N2, and N3. The winged helix transcription factor HNF3 binds to two sites near the center of the N1 particle. We created dinucleosome templates using the albumin enhancer sequence and found that site-specific binding of HNF3 protein resulted in nucleosome positioning in vitro similar to that seen in liver nuclei. Thus, binding of a transcription factor can position an underlying nucleosome core.

Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Allosteric interaction of a herpes simplex viral thymidine kinase with host DNA polymerase alpha in mouse LP1-1 cells.

A DNA polymerase alpha-associated multienzyme complex isolated from mouse LP1-1 cells transfected with the thymidine kinase gene of herpes simplex virus type I (1) showed activities of DNA polymerase alpha, topoisomerase II, and thymidine kinase (TK) in the complex. TK antiserum recognized a 43 kDa polypeptide only in the fraction of the multienzyme complex prepared from the LP1-1 cells but not that from L-M(TK-) cells. In permeabilized cells, hydroxyurea did not show any inhibitory effect on either DNA polymerase or TK, whereas aphidicolin, novobiocin, and TK antiserum inhibited both enzymes. These results provide evidence for the functional association and an allosteric interaction between the viral TK and host DNA polymerase alpha.

An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array.

Nucleosomes positioned over promoters are usually inhibitory to protein binding and activity. We analyzed at the nucleotide level of resolution the nucleosomal organization of a distal, liver-specific enhancer in various mouse tissues and found that the enhancer exists in an array of three precisely positioned nucleosomes only in liver chromatin, where the enhancer is active. In vivo footprinting reveals that essential transcription factor-binding sites are occupied on apparent nucleosome surfaces, in one case leading to a perturbed nucleosomal structure. A similar nucleosomal array is generated with an in vitro chromatin assembly system in which nucleosome positioning is dependent upon binding to the enhancer of proteins related to hepatocyte nuclear factor 3. We suggest that certain transcription factors can organize nucleosomal structures that define an active enhancer element.