Computational Studies on Selected Macrolides Active against Escherichia coli Combined with the NMR Study of Tylosin A in Deuterated Chloroform

Abstract: Although many antibiotics are active against Gram-positive bacteria, fewer also show activity against Gram-negative bacteria. Here, we present a combination of in silico (electron ion-interaction potential, molecular docking, ADMET), NMR, and microbiological investigations of selected macrolides (14-membered, 15-membered, and 16-membered), aiming to discover the pattern of design for macrolides active against Gram-negative bacteria. Although the conforma-tional studies of 14-membered and 15-membered macrolides are abundant in the literature, 16-membered macrolides, and their most prominent representative tylosin A, have received rela-tively little research attention. We therefore report the complete 1H and 13C NMR assignment of tylosin A in deuterated chloroform, as well as its 3D solution structure determined through mo-lecular modelling (conformational search) and 2D ROESY NMR. Additionally, due to the degra-dation of tylosin A in deuterated chloroform, other species were also detected in 1D and 2D NMR spectra. We additionally studied the anti-bacterial activity of tylosin A and B against se-lected Gram-positive and Gram-negative bacteria

N-glycosylation and expression in human tissues of the orphan GPR61 receptor

A number of members of the G protein-coupled receptor class of cell surface receptors are 'orphans' with no known endogenous ligand. One of these orphan receptors is GPR61; there are little data about its expression in human cells and tissues. In this study, we investigated the post-translational modification of GPR61 by N-glycosylation at an identified consensus N-glycosylation site (N12) and the impact of this modification upon the subcellular expression of the protein. The N-glycosylation inhibitor tunicamycin reduced the apparent molecular weight of immunoreactivity associated with myc-tagged GPR61 by 1-2 kDa, which was comparable to the evident molecular weight of the myc-tagged N12S GPR61 mutant with disrupted consensus N-glycosylation site. Analysis of GPR61 expression demonstrated that tunicamycin treatment reduced considerably heterologous expression of GPR61 in the cell membrane despite the N12S GPR61 mutant being readily expressed at the cell surface. These results demonstrate that GPR61 is subject to N-glycosylation but suggest this is not a prerequisite for cell surface expression, although N-glycosylation of other proteins may be important for cell membrane expression of GPR61. Expression of GPR61 protein was demonstrated at the cellular level in human hippocampus and human peripheral blood mononuclear cells. In the latter, there was a significantly higher expression of GPR61 in the Th17 cell subset in comparison with resting CD4+ cells, which may point toward a potential role for the GPR61 receptor in autoimmune diseases. This is the first report that GPR61 protein is subject to post-translational modification and is expressed in immune cell subsets and the hippocampus. These findings will help guide studies to investigate the function of GPR61

A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data

Background: Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle (Cq) standard curve quantification, which requires the time- and labor-intensive construction of a Cq standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as Cq standard curve quantification. Principal Findings: We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as Cq standard curve quantification for a variety of DNA targets and a wide range of concentrations. Significance: We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sampl

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample

Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, −2.1%, and −13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA

Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p><it>Entamoeba histolytica </it>is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in <it>Entamoeba histolytica</it>.</p> <p>Results</p> <p>An episomal vector-based system, using the <it>E. histolytica </it>U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in <it>E. histolytica</it>. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%.</p> <p>Conclusion</p> <p>Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in <it>Entamoeba histolytica</it>, providing a useful tool for the study of this parasite.</p

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays.We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation

Biogenesis of mammalian microRNAs by a non-canonical processing pathway

Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed ‘simtrons’, does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs

Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells

MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease