Enhancer cooperativity can compensate for loss of activity over large genomic distances.

Enhancers are short DNA sequences that activate their target promoter from a distance; however, increasing the genomic distance between the enhancer and the promoter decreases expression levels. Many genes are controlled by combinations of multiple enhancers, yet the interaction and cooperation of individual enhancer elements are not well understood. Here, we developed a synthetic platform in mouse embryonic stem cells that allows building complex regulatory landscapes from the bottom up.

Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection.

Stem cells intrinsically express a subset of genes which are normally associated with interferon stimulation and the innate immune response. However, the expression of these interferon-stimulated genes (ISG) in stem cells is independent from external stimuli such as viral infection. Here, we show that the interferon regulatory factor 1, Irf1, is directly controlled by the murine formative pluripotency gene regulatory network and transiently upregulated during the transition from naive to formative pluripotency.

Omega-3 Index and Obstructive Sleep Apnea: A Cross-Sectional Study.

Study Objectives: Erythrocyte levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Omega-3 Index) were previously found to be associated with obstructive sleep apnea (OSA) at very low levels (< 5.0%) in only one epidemiologic study. OSA has comorbidities, such as arterial hypertension, heart failure, or major depression, also associated with a low Omega-3 Index.

A longitudinal proteomic assessment of peptide degradation and loss under acidic storage conditions.

Sample preparation prior to analysis by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) usually involves the storage of frozen peptide samples in an acidic environment for variable time periods. Questions arose in our laboratory regarding the stability of peptides in acid under medium- to long-term storage. Thus, a 10-month longitudinal study was designed to assess the effect on storage of tryptic peptides at -20 and -80°C under acidic conditions.

abFASP-MS: affinity-based filter-aided sample preparation mass spectrometry for quantitative analysis of chemically labeled protein complexes.

Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC-MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE.

Multiple and sequential data acquisition method: an improved method for fragmentation and detection of cross-linked peptides on a hybrid linear trap quadrupole Orbitrap Velos mass spectrometer.

The identification and validation of cross-linked peptides by mass spectrometry remains a daunting challenge for protein-protein cross-linking approaches when investigating protein interactions. This includes the fragmentation of cross-linked peptides in the mass spectrometer per se and following database searching, the matching of the molecular masses of the fragment ions to the correct cross-linked peptides. The hybrid linear trap quadrupole (LTQ) Orbitrap Velos combines the speed of the tandem mass spectrometry (MS/MS) duty circle with high mass accuracy, and these features were utilized in the current study to substantially improve the confidence in the identification of cross-linked peptides.

A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.

Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis.

Combining filter-aided sample preparation and pseudoshotgun technology to profile the proteome of a low number of early passage human melanoma cells.

The performance of two proteomic sample preparation methods, "pseudoshotgun" (PSG) and filter-aided sample preparation (FASP) were compared in terms of the number of identified proteins, representation of cellular component GO (gene ontology) categories in the obtained list of proteins, and the efficiency of both methods in the proteomic analysis of a very low number of cells. Both methods were combined to obtain a proteomic profile of a short-term culture (passage 3) of melanoma cells, established in our laboratory from a human metastatic melanoma lesion. The data revealed that with FASP, usually more proteins are identified than with PSG when analyzing a higher number of cells (≥ 5000/injection), whereas PSG is favorable when analyzing only a very small amount of cells (250-500/injection).

Mass spectrometric characterization of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT(1A) R) expressed in tsA201 human embryonic kidney cells.

The 5-hydroxytryptamine 1A receptor (serotonin 1A receptor; 5-HT(1A) R) is involved in a large series of brain functions, and roles in anxiety, depression, and cognition have been reported. So far, published information on mass spectrometrical characterization of 5-HT(1A) R is limited to the presence of two 5-HT(1A) R peptides in rat's whole brain as observed by in-solution digestion followed by LC-MS/MS. Knowledge about the protein sequence and PTMs, however, would have implications for generation of specific antibodies and designing studies on the 5-HT(1A) R at the protein level.

Interdisciplinary critique of sipuleucel-T as immunotherapy in castration-resistant prostate cancer.

Sipuleucel-T was approved by the US Food and Drug Administration on April 29, 2010, as an immunotherapy for late-stage prostate cancer. To manufacture sipuleucel-T, mononuclear cells harvested from the patient are incubated with a recombinant prostatic acid phosphatase (PAP) antigen and reinfused. The manufacturer proposes that antigen-presenting cells exogenously activated by PAP induce endogenous T-cells to attack PAP-bearing prostate cancer cells.