The Rich Mid-Infrared Environments of Two Highly-Obscured X-ray Binaries: Spitzer Observations of IGR J16318-4848 and GX 301-2

We present the results of Spitzer mid-infrared spectroscopic observations of two highly-obscured massive X-ray binaries: IGR J16318-4848 and GX301-2. Our observations reveal for the first time the extremely rich mid-infrared environments of this type of source, including multiple continuum emission components (a hot component with T > 700 K and a warm component with T ~ 180 K) with apparent silicate absorption features, numerous HI recombination lines, many forbidden ionic lines of low ionization potentials, and pure rotational H2 lines. This indicates that both sources have hot and warm circumstellar dust, ionized stellar winds, extended low-density ionized regions, and photo-dissociated regions. It appears difficult to attribute the total optical extinction of both sources to the hot and warm dust components, which suggests that there could be an otherwise observable colder dust component responsible for the most of the optical extinction and silicate absorption features. The observed mid-infrared spectra are similar to those from Luminous Blue Variables, indicating that the highly-obscured massive X-ray binaries may represent a previously unknown evolutionary phase of X-ray binaries with early-type optical companions. Our results highlight the importance and utility of mid-infrared spectroscopy to investigate highly-obscured X-ray binaries.Comment: To appear in ApJ Letter

Highly Parallel Genome-Wide Expression Analysis of Single Mammalian Cells

We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology

Regulation of Hypoxia-Inducible Factor 1α Expression and Function by the Mammalian Target of Rapamycin

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1α subunit and a constititutively expressed HIF-1β subunit. Under hypoxic conditions, the HIF-1α subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1α-HIF-1β heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl(2). Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1α and HIF-1-dependent transcription induced by hypoxia or CoCl(2). Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl(2), while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1α stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1α fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1α stabilization by CoCl(2). These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress

Quantitative phenotyping via deep barcode sequencing

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or “Bar-seq,” outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that ∼20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene–environment interactions on a genome-wide scale

The average relative quantities of mtDNA observed in association with female age at the cleavage stage.

<p>The mtDNA values (2-^{Delta Delta Ct}) were obtained during real-time PCR analysis. All examined blastomeres were characterised as being chromosomally normal.</p><p>The average relative quantities of mtDNA observed in association with female age at the cleavage stage.</p

The mtDNA content of chromosomally normal blastocysts in relation to clinical outcome.

<p>On average, chromosomally normal blastocysts capable of establishing a clinical pregnancy contained significantly (P = 0.007) lower levels of mtDNA compared to chromosomally normal blastocysts that failed to do so.</p

Blastocyst mtDNA quantity threshold in relation to clinical outcome.

<p><b>a)</b> The mtDNA quantity viability threshold for euploid blastocysts, established via retrospective analysis of TE biopsies from transferred embryos with known outcomes. All blastocysts producing viable pregnancies contained mtDNA quantities below the 0.003 value (red line) whereas mtDNA quantities above this value were associated with failure to achieve an ongoing clinical pregnancy. <b>b)</b> Results of the prospective blinded study. The mtDNA threshold used was the same as that established in the retrospective study (4a). Validity was confirmed, since all blastocysts producing viable pregnancies contained mtDNA quantities below the cut-off (red line) and no blastocysts with mtDNA quantities above this value achieved an ongoing clinical pregnancy. <b>c)</b> NGS analysis of the mtDNA level in 23 euploid TE samples. The corresponding embryos were transferred during SET cycles, and clinical outcomes were known for 21 of them. As with the real-time PCR experiments, mtDNA levels were lower in the seven implanting embryos (note- the y-axis scale is different for NGS analyses and consequently cut-off values differ).</p