The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA–protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.     

Identification and Characterization of Propionylation at Histone H3 Lysine 23 in Mammalian Cells

Propionylation has been identified recently as a new type of protein post-translational modification. Bacterial propionyl-CoA synthetase and human histone H4 are propionylated at specific lysine residues that have been known previously to be acetylated. However, other proteins subject to this modification remain to be identified, and the modifying enzymes involved need to be characterized. In this work, we report the discovery of histone H3 propionylation in mammalian cells. Propionylation at H3 lysine Lys²³ was detected in the leukemia cell line U937 by mass spectrometry and Western analysis using a specific antibody. In this cell line, the propionylated form of Lys²³ accounted for 7%, a level at least 6-fold higher than in other leukemia cell lines (HL-60 and THP-1) or non-leukemia cell lines (HeLa and IMR-90). The propionylation level in U937 cells decreased remarkably during monocytic differentiation, indicating that this modification is dynamically regulated. Moreover, in vitro assays demonstrated that histone acetyltransferase p300 can catalyze H3 Lys²³ propionylation, whereas histone deacetylase Sir2 can remove this modification in the presence of NAD⁺. These results suggest that histone propionylation might be generated by the same set of enzymes as for histone acetylation and that selection of donor molecules (propionyl-CoA versus acetyl-CoA) may determine the difference of modifications. Because like acetyl-CoA, propionyl-CoA is an important intermediate in biosynthesis and energy production, histone H3 Lys²³ propionylation may provide a novel epigenetic regulatory mark for cell metabolism.     

Bat point counts: A novel sampling method shines light on flying bat communities

Emerging technologies based on the detection of electro‐magnetic energy offer promising opportunities for sampling biodiversity. We exploit their potential by showing here how they can be used in bat point counts—a novel method to sample flying bats—to overcome shortcomings of traditional sampling methods, and to maximize sampling coverage and taxonomic resolution of this elusive taxon with minimal sampling bias. We conducted bat point counts with a sampling rig combining a thermal scope to detect bats, an ultrasound recorder to obtain echolocation calls, and a near‐infrared camera to capture bat morphology. We identified bats with a dedicated identification key combining acoustic and morphological features, and compared bat point counts with the standard bat sampling methods of mist‐netting and automated ultrasound recording in three oil palm plantation sites in Indonesia, over nine survey nights. Based on rarefaction and extrapolation sampling curves, bat point counts were similarly effective but more time‐efficient than the established methods for sampling the oil palm species pool in our study. Point counts sampled species that tend to avoid nets and those that are not echolocating, and thus cannot be detected acoustically. We identified some bat sonotypes with near‐infrared imagery, and bat point counts revealed strong sampling biases in previous studies using capture‐based methods, suggesting similar biases in other regions might exist. Our method should be tested in a wider range of habitats and regions to assess its performance. However, while capture‐based methods allow to identify bats with absolute and internal morphometry, and unattended ultrasound recorders can effectively sample echolocating bats, bat point counts are a promising, non‐invasive, and potentially competitive new tool for sampling all flying bats without bias and observing their behavior in the wild.     

Optimal Tracking Controller Design for a Small Scale Helicopter

A model helicopter is more difficult to control than its full scale counterpart. This is due to its greater sensitivity to control inputs and disturbances as well as higher bandwidth of dynamics. This work is focused on designing practical tracking controller for a small scale helicopter following predefined trajectories. A tracking controller based on optimal control theory is synthesized as a part of the development of an autonomous helicopter. Some issues with regards to control constraints are addressed.The weighting between state tracking performance and control power expenditure is analyzed. Overall performance of the control design is evaluated based on its time domain histories of trajectories as well as control inputs.     

Clinical and Cost-Effectiveness of Procalcitonin Test for Prodromal Meningococcal Disease–A Meta-Analysis

Background

Despite vaccines and improved medical intensive care, clinicians must continue to be vigilant of possible Meningococcal Disease in children. The objective was to establish if the procalcitonin test was a cost-effective adjunct for prodromal Meningococcal Disease in children presenting at emergency department with fever without source.

Methods and Findings

Data to evaluate procalcitonin, C-reactive protein and white cell count tests as indicators of Meningococcal Disease were collected from six independent studies identified through a systematic literature search, applying PRISMA guidelines. The data included 881 children with fever without source in developed countries.The optimal cut-off value for the procalcitonin, C-reactive protein and white cell count tests, each as an indicator of Meningococcal Disease, was determined. Summary Receiver Operator Curve analysis determined the overall diagnostic performance of each test with 95% confidence intervals. A decision analytic model was designed to reflect realistic clinical pathways for a child presenting with fever without source by comparing two diagnostic strategies: standard testing using combined C-reactive protein and white cell count tests compared to standard testing plus procalcitonin test. The costs of each of the four diagnosis groups (true positive, false negative, true negative and false positive) were assessed from a National Health Service payer perspective. The procalcitonin test was more accurate (sensitivity=0.89, 95%CI=0.76-0.96; specificity=0.74, 95%CI=0.4-0.92) for early Meningococcal Disease compared to standard testing alone (sensitivity=0.47, 95%CI=0.32-0.62; specificity=0.8, 95% CI=0.64-0.9). Decision analytic model outcomes indicated that the incremental cost effectiveness ratio for the base case was £-8,137.25 (US $ -13,371.94) per correctly treated patient.

Conclusions

Procalcitonin plus standard recommended tests, improved the discriminatory ability for fatal Meningococcal Disease and was more cost-effective; it was also a superior biomarker in infants. Further research is recommended for point-of-care procalcitonin testing and Markov modelling to incorporate cost per QALY with a life-time model.     

Complement component 1, q subcomponent binding protein is a marker for proliferation in breast cancer

Complement component 1, q subcomponent binding protein (C1QBP), is a multi-compartmental protein with higher mRNA expression reported in breast cancer tissues. This study evaluated the association between immunohistochemical expression of the C1QBP protein in breast cancer tissue microarrays (TMAs) and clinicopathological parameters, in particular tumor size. In addition, an in vitro study was conducted to substantiate the breast cancer TMA findings. Breast cancer TMAs were constructed from pathological specimens of patients diagnosed with invasive ductal carcinoma. C1QBP protein and proliferating cell nuclear antigen (PCNA) immunohistochemical analyses were subsequently performed in the TMAs. C1QBP immunostaining was detected in 131 out of 132 samples examined. The C1QBP protein was predominantly localized in the cytoplasm of the breast cancer cells. Univariate analysis revealed that a higher C1QBP protein expression was significantly associated with older patients (P = 0.001) and increased tumor size (P = 0.002). Multivariate analysis showed that C1QBP is an independent predictor of tumor size in progesterone-positive tumors. Furthermore, C1QBP was also significantly correlated with expression of PCNA, a known marker of proliferation. Inhibition of C1QBP expression was performed by transfecting C1QBP siRNA into T47D breast cancer cells, a progesterone receptor-positive breast cancer cell line. C1QBP gene expression was analyzed by real-time RT-PCR, and protein expression by Western blot. Cell proliferation assays were also performed by commercially available assays. Down-regulation of C1QBP expression significantly decreased cell proliferation and growth in T47D cells. Taken together, our findings suggest that the C1QBP protein could be a potential proliferative marker in breast cancer.     

Impact of protein stability, cellular localization, and abundance on proteomic detection of tumor-derived proteins in plasma

Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the most abundant intracellular proteins.     

Mechanisms of Base Selection by Human Single-stranded Selective Monofunctional Uracil-DNA Glycosylase

hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved.Three glycosylases that initiate DNA repair via the base excision repair (BER)2 pathway are found on human chromosome 12. These three glycosylases are designated as UNG, TDG, and hSMUG1 (1–4). Several groups are currently investigating the structure and properties of these glycosylases in order to determine their physiological roles. A common property of these enzymes is the cleavage of uracil residues from DNA, although each of the glycosylases repairs additional lesions. Despite low sequence homology (8%), these three glycosylases share a common fold and overall architecture (5). Subtle differences in structure apparently distinguish these repair enzymes with respect to substrate and context preferences.UNG is the most active of the glycosylases. UNG recognizes uracil residues when found in single strand, or double strand DNA paired with adenine or mispaired with guanine (6); however, only a small number of other pyrimidines are also targets. UNG is spliced into two forms, UNG1 and UNG2. UNG1 is targeted to the mitochondrion, whereas UNG2 is found primarily in the cell nucleus (7). Due to the capacity of UNG to repair uracil in many contexts, as well as its association with DNA replication machinery and cell cycle specificity, it is thought that a primary role for UNG is in the repair of uracil misincorporated opposite adenine during DNA replication (8, 9). Recent studies also suggest an important role for UNG in removing uracil residues in DNA generated by activation-induced deaminase as part of somatic hypermutation and class switch recombination in activated B-cells (10–12).In contrast to UNG, the related glycosylases hSMUG1 and TDG appear to target uracil and uracil analogs mispaired with guanine (3, 13, 14). Although hSMUG1 was originally characterized as a single strand selective glycosylase (13), more recent studies suggest it is more active on mispaired uracil in duplex DNA (14), and it has an extended substrate range, removing several oxidized pyrimidines (15–18), including 5-hydroxymethyluracil (HmU), 5-formyluracil (FoU), and 5-hydroxyuracil (HoU). TDG appears to act exclusively on duplex substrates, with a strong preference for mispaired pyrimidines, including thymine, and a strong preference for damage located in CpG dinucleotides (19–21). The apparent sequence selectivity of TDG has led to suggestions that the primary role of TDG is the repair of deaminated 5-methylcytosine residues in CpG dinucleotides (20).In this paper, we have investigated the enzymatic properties of recombinant human SMUG1 in single-turnover kinetic assays on a series of oligonucleotide substrates containing purine and pyrimidine analogs. In the first set of experiments, the capacity of hSMUG1 to cleave uracil opposite a series of purine analogs was measured to determine if the preference of hSMUG1 for mispairs can be attributed to reduced duplex stability or if hSMUG1 recognizes specific functional groups on the purine opposite the target uracil. In the second series of experiments, a series of 5-substituted uracil analogs was paired opposite guanine to probe the mechanisms by which hSMUG1 distinguishes potential substrates. This series includes uracil, a series of oxidatively damaged pyrimidines, and the 5-halouracils, which serve to measure both substituent size and electronic inductive properties. New to this series is 5-carboxyuracil (CaU), the last in the sequence of damage products arising from oxidation of the thymine methyl group (22–24).Previous studies with other glycosylases described above have highlighted the importance of size and electronic inductive properties of 5-substituted pyrimidines in substrate selection. In contrast, the capacity of hSMUG1 to recognize HmU but not thymine has been attributed to the hydrophilicity and hydrogen-bonding capacity of the HmU substituent (15–18). In this paper, selected physical properties have been calculated for each pyrimidine examined, including solvent-accessible surface area (SASA) and the free energy of solvation in water. The SASA is introduced as a parameter to define the relative size of the 5-substituted pyrimidines, whereas the free energy of solvation in water is proposed to describe the capacity of the 5-substituted pyrimidine to interact with or replace water within the hSMUG1 pyrimidine binding pocket. The observed kinetic rate constants are compared with the physical properties of the modified bases and base pairs in order to explain the mechanisms by which hSMUG1 identifies and distinguishes target lesions. Our results indicate that the strategies used by hSMUG1 to select target bases and avoid normal bases contrast with those of other members of the uracil DNA-glycosylase family.