About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra-loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs.
We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.
We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)
Purpose: Patients with HER2-positive metastatic breast cancer (MBC) with central nervous system (CNS) metastasis have a poor prognosis. We report treatments and outcomes in patients with HER2-positive MBC and CNS metastasis from the Systemic Therapies for HER2-positive Metastatic Breast Cancer Study (SystHERs). Experimental Design: SystHERs (NCT01615068) was a prospective, U.S.-based, observational registry of patients with newly diagnosed HER2-positive MBC. Study endpoints included treatment patterns, clinical outcomes, and patientreported outcomes (PRO). Results: Among 977 eligible patients enrolled (2012-2016), CNS metastasis was observed in 87 (8.9%) at initial MBC diagnosis and 212 (21.7%) after diagnosis, and was not observed in 678 (69.4%) patients. White and younger patients, and those with recurrent MBC and hormone receptor negative disease, had higher risk of CNS metastasis. Patients with CNS metastasis at diagnosis received first-line lapatinib more commonly (23.0% vs. 2.5%), and trastuzumab less commonly (70.1% vs. 92.8%), than patients without CNS metastasis at diagnosis. Risk of death was higher with CNS metastasis observed at or after diagnosis [median overall survival (OS) 30.2 and 38.3 months from MBC diagnosis, respectively] versus no CNS metastasis [median OS not estimable: HR 2.86; 95% confidence interval (CI), 2.05-4.00 and HR 1.94; 95% CI, 1.52-2.49]. Patients with versus without CNS metastasis at diagnosis had lower quality of life at enrollment. Conclusions: Despite advances in HER2-targeted treatments, patients with CNS metastasis continue to have a poor prognosis and impaired quality of life. Observation of CNS metastasis appears to influence HER2-targeted treatment choice.
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