Input DNA Ratio Determines Copy Number of The 33 kb Factor IX Gene on De Novo Human Artificial Chromosomes

Human artificial chromosomes (ACs) are non-integrating vectors that may be useful for gene therapy. They assemble in cultured cells following transfection of human centromeric α -satellite DNA and segregate efficiently alongside the host genome. In the present study, a 33 kilobase (kb) Factor IX (FIX) gene was incorporated into mitotically stable ACs in human HT1080 lung derived cells using co-transfection of a bacterial artificial chromosome (BAC) harboring synthetic α -satellite DNA and a P1 artificial chromosome(PAC) that spans the FIX locus. ACs were detected in ≥90% of chromosome spreads in 8 of 19 lines expanded from drug resistant colonies. FIX transgene copy number on ACs was determined by input DNA transfection ratios. Furthermore, a low level of FIX transcription was detected from ACs with multiple transgenes but not from those incorporating a single transgene, suggesting that reducing transgene number may limit misexpression. Their potential to segregate cross species was measured by transferring ACs into mouse and hamster cell lines using microcell-mediated chromosome transfer. Lines were obtained where ACs segregated efficiently. The stable segregation of ACs in rodent cells suggests that it should be possible to develop animal models to test the capacity of ACs to rescue FIX deficiency

The 74MHz System on the Very Large Array

The Naval Research Laboratory and the National Radio Astronomy Observatory completed implementation of a low frequency capability on the VLA at 73.8 MHz in 1998. This frequency band offers unprecedented sensitivity (~25 mJy/beam) and resolution (~25 arcsec) for low-frequency observations. We review the hardware, the calibration and imaging strategies, comparing them to those at higher frequencies, including aspects of interference excision and wide-field imaging. Ionospheric phase fluctuations pose the major difficulty in calibrating the array. Over restricted fields of view or at times of extremely quiescent ionospheric ``weather'', an angle-invariant calibration strategy can be used. In this approach a single phase correction is devised for each antenna, typically via self-calibration. Over larger fields of view or at times of more normal ionospheric ``weather'' when the ionospheric isoplanatic patch size is smaller than the field of view, we adopt a field-based strategy in which the phase correction depends upon location within the field of view. This second calibration strategy was implemented by modeling the ionosphere above the array using Zernike polynomials. Images of 3C sources of moderate strength are provided as examples of routine, angle-invariant calibration and imaging. Flux density measurements indicate that the 74 MHz flux scale at the VLA is stable to a few percent, and tied to the Baars et al. value of Cygnus A at the 5 percent level. We also present an example of a wide-field image, devoid of bright objects and containing hundreds of weaker sources, constructed from the field-based calibration. We close with a summary of lessons the 74 MHz system offers as a model for new and developing low-frequency telescopes. (Abridged)Comment: 73 pages, 46 jpeg figures, to appear in ApJ

Selective inhibition of pancreatic ductal adenocarcinoma cell growth by the mitotic MPS1 kinase inhibitor NMS-P715

Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy

Radio Frequency Spectra of 388 Bright 74 MHz Sources

As a service to the community, we have compiled radio frequency spectra from the literature for all sources within the VLA Low Frequency Sky Survey (VLSS) that are brighter than 15 Jy at 74 MHz. Over 160 references were used to maximize the amount of spectral data used in the compilation of the spectra, while also taking care to determine the corrections needed to put the flux densities from all reference on the same absolute flux density scale. With the new VLSS data, we are able to vastly improve upon previous efforts to compile spectra of bright radio sources to frequencies below 100 MHz because (1) the VLSS flux densities are more reliable than those from some previous low frequency surveys and (2) the VLSS covers a much larger area of the sky (declination >-30 deg.) than many other low frequency surveys (e.g., the 8C survey). In this paper, we discuss how the spectra were constructed and how parameters quantifying the shapes of the spectra were derived. Both the spectra and the shape parameters are made available here to assist in the calibration of observations made with current and future low frequency radio facilities.Comment: Accepted to ApJ

An inhibitor of the mitotic kinase, MPS1, is selective towards pancreatic cancer cells

poster abstractThe abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer

LOFAR 150-MHz observations of SS 433 and W50

We present Low-Frequency Array (LOFAR) high-band data over the frequency range 115-189 MHz for the X-ray binary SS 433, obtained in an observing campaign from 2013 February to 2014 May. Our results include a deep, wide-field map, allowing a detailed view of the surrounding supernova remnant W50 at low radio frequencies, as well as a light curve for SS 433 determined from shorter monitoring runs. The complex morphology of W50 is in excellent agreement with previously published higher frequency maps; we find additional evidence for a spectral turnover in the eastern wing, potentially due to foreground free-free absorption. Furthermore, SS 433 is tentatively variable at 150 MHz, with both a debiased modulation index of 11 per cent and a Χ 2 probability of a flat light curve of 8.2 × 10 -3 . By comparing the LOFAR flux densities with contemporaneous observations carried out at 4800 MHz with the RATAN-600 telescope, we suggest that an observed ~0.5-1 Jy rise in the 150-MHz flux density may correspond to sustained flaring activity over a period of approximately 6 months at 4800 MHz. However, the increase is too large to be explained with a standard synchrotron bubble model. We also detect a wealth of structure along the nearby Galactic plane, including the most complete detection to date of the radio shell of the candidate supernova remnant G38.7-1.4. This further demonstrates the potential of supernova remnant studies with the current generation of low-frequency radio telescopes

Input DNA Ratio Determines Copy Number of The 33 kb Factor IX Gene on De Novo Human Artificial Chromosomes

Human artificial chromosomes (ACs) are non-integrating vectors that may be useful for gene therapy. They assemble in cultured cells following transfection of human centromeric α -satellite DNA and segregate efficiently alongside the host genome. In the present study, a 33 kilobase (kb) Factor IX (FIX) gene was incorporated into mitotically stable ACs in human HT1080 lung derived cells using co-transfection of a bacterial artificial chromosome (BAC) harboring synthetic α -satellite DNA and a P1 artificial chromosome(PAC) that spans the FIX locus. ACs were detected in ≥90% of chromosome spreads in 8 of 19 lines expanded from drug resistant colonies. FIX transgene copy number on ACs was determined by input DNA transfection ratios. Furthermore, a low level of FIX transcription was detected from ACs with multiple transgenes but not from those incorporating a single transgene, suggesting that reducing transgene number may limit misexpression. Their potential to segregate cross species was measured by transferring ACs into mouse and hamster cell lines using microcell-mediated chromosome transfer. Lines were obtained where ACs segregated efficiently. The stable segregation of ACs in rodent cells suggests that it should be possible to develop animal models to test the capacity of ACs to rescue FIX deficiency

“ASSESSMENT OF CHROMOSOME INSTABILITY IN TRIPLE-NEGATIVE BREAST CANCERS USING NUCLEI HARVESTED“ASSESSMENT OF CHROMOSOME INSTABILITY IN TRIPLE-NEGATIVE BREAST CANCERS USING NUCLEI HARVESTED FROM FROZEN TISSUES” FROM FROZEN TISSUES”

poster abstractChromosomal instability (CIN), defined as ongoing chromosome mis-segregation, is prevalent in the majority of solid tumors and potentially contributes to cancer progression and hazardous genetic changes. Optimization of a common laboratory technique to assess CIN in isolated nuclei will benefit basic research and possibly be useful for clinical diagnostic purposes. Preliminary studies have demonstrated a successful protocol for performing fluorescence in situ hybridization (FISH) on nuclei harvested from frozen tumor and normal breast tissues. The frozen breast tumors were of the triple-negative breast cancer (TNBC) sub-type that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor-2 (HER2). Six TNBCs analyzed to date (20-50 nuclei per tumor) exhibited chromosome instability using centromere specific probes in FISH analysis. Modal centromere number deviation (MCD)/sample was used to calculate CIN levels. Percent MCD ranged from 32-68% in TNBCs and contrasted with the normal breast tissue sample that exhibited 2% MCD. Previous FISH studies on tissue sections by others have shown that ER negative breast tumors with greater than 45% MCD had a better prognosis. Further study will be required to determine whether CIN levels (measured by MCD) can serve as a biomarker for stratifying TNBC patients into likely responders and non- responders to treatment. Chromatin immunoprecipitation (ChIP) assays performed in parallel from the same frozen tissue revealed that centromeric heterochromatin structure is altered in TNBCs and may contribute to chromosome instability. The ability to perform both FISH and ChIP analysis on frozen human breast tissue has provided a foundation for further exploration of the relationship between CIN and centromere malfunction in tumor tissues and opens up therapeutic possibilities targeting the CIN phenotype in TNBCs

An inhibitor of the mitotic kinase, MPS1, is selective towards pancreatic cancer cells

poster abstractThe abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer