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101.
Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood
Al-Salmani K Abbas HH Schulpen S Karbaschi M Abdalla I Bowman KJ So KK Evans MD Jones GD Godschalk RW Cooke MS 《Free radical biology & medicine》2011,51(3):719-725
Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed. 相似文献
102.
Significant cell damage occurs during cryopreservation resulting in a decreased number of viable and functional cells post-thawing. Recent studies have correlated the unsuccessful outcome of regenerative therapies with poor cell viability after cryopreservation. Cell damage from ice recrystallization during freeze-thawing is one cause of decreased viability after cryopreservation. We have assessed the ability of two C-AFGPs that are potent inhibitors of ice recrystallization to increase cell viability after cryopreservation. Our results indicate that a 1-1.5 mg/mL (0.5-0.8 mM) solution of C-AFGP 1 is an excellent alternative to a 2.5% DMSO solution for the cryopreservation of human embryonic liver cells. 相似文献
103.
Mi-Youn K Brusniak Sung-Tat Kwok Mark Christiansen David Campbell Lukas Reiter Paola Picotti Ulrike Kusebauch Hector Ramos Eric W Deutsch Jingchun Chen Robert L Moritz Ruedi Aebersold 《BMC bioinformatics》2011,12(1):1-15
Background
Copy number variants (CNVs), including deletions, amplifications, and other rearrangements, are common in human and cancer genomes. Copy number data from array comparative genome hybridization (aCGH) and next-generation DNA sequencing is widely used to measure copy number variants. Comparison of copy number data from multiple individuals reveals recurrent variants. Typically, the interior of a recurrent CNV is examined for genes or other loci associated with a phenotype. However, in some cases, such as gene truncations and fusion genes, the target of variant lies at the boundary of the variant.Results
We introduce Neighborhood Breakpoint Conservation (NBC), an algorithm for identifying rearrangement breakpoints that are highly conserved at the same locus in multiple individuals. NBC detects recurrent breakpoints at varying levels of resolution, including breakpoints whose location is exactly conserved and breakpoints whose location varies within a gene. NBC also identifies pairs of recurrent breakpoints such as those that result from fusion genes. We apply NBC to aCGH data from 36 primary prostate tumors and identify 12 novel rearrangements, one of which is the well-known TMPRSS2-ERG fusion gene. We also apply NBC to 227 glioblastoma tumors and predict 93 novel rearrangements which we further classify as gene truncations, germline structural variants, and fusion genes. A number of these variants involve the protein phosphatase PTPN12 suggesting that deregulation of PTPN12, via a variety of rearrangements, is common in glioblastoma.Conclusions
We demonstrate that NBC is useful for detection of recurrent breakpoints resulting from copy number variants or other structural variants, and in particular identifies recurrent breakpoints that result in gene truncations or fusion genes. Software is available at http://http.//cs.brown.edu/people/braphael/software.html. 相似文献104.
105.
Background
The key epidemiological difference between pandemic and seasonal influenza is that the population is largely susceptible during a pandemic, whereas, during non-pandemic seasons a level of immunity exists. The population-level efficacy of household-based mitigation strategies depends on the proportion of infections that occur within households. In general, mitigation measures such as isolation and quarantine are more effective at the population level if the proportion of household transmission is low.Methods/Results
We calculated the proportion of infections within households during pandemic years compared with non-pandemic years using a deterministic model of household transmission in which all combinations of household size and individual infection states were enumerated explicitly. We found that the proportion of infections that occur within households was only partially influenced by the hazard h of infection within household relative to the hazard of infection outside the household, especially for small basic reproductive numbers. During pandemics, the number of within-household infections was lower than one might expect for a given because many of the susceptible individuals were infected from the community and the number of susceptible individuals within household was thus depleted rapidly. In addition, we found that for the value of at which 30% of infections occur within households during non-pandemic years, a similar 31% of infections occur within households during pandemic years.Interpretation
We suggest that a trade off between the community force of infection and the number of susceptible individuals in a household explains an apparent invariance in the proportion of infections that occur in households in our model. During a pandemic, although there are more susceptible individuals in a household, the community force of infection is very high. However, during non-pandemic years, the force of infection is much lower but there are fewer susceptible individuals within the household. 相似文献106.
Endothelin‐1 (ET‐1) has been demonstrated to induce insulin resistance (IR) and lipolysis, raising the possibility that ET‐1 may also contribute to the elevated fatty acid levels in IR‐associated comorbidities. We attempted to evaluate whether ET‐1 also affects the long‐chain fatty acid (LCFA) utilization in 3T3‐L1 adipocytes. The effects of chronic ET‐1 exposure on basal and insulin‐stimulated LCFA uptake, and LCFA uptake kinetics were examined in 3T3‐L1 adipocytes. Chronic exposure to ET‐1 induced IR and suppressed basal and insulin‐stimulated LCFA uptake. Given that insulin acutely stimulates LCFA uptake, there was dramatically similar trend of dose‐response curves for ET‐1‐suppressed LCFA uptake, and also similar corresponding IC50 values, between basal and insulin‐stimulated states, reflecting that ET‐1 predominantly suppresses basal LCFA uptake. Results of LCFA kinetics, western blots, and CD36 inhibition using sulfosuccinimidyl oleate (SSO) revealed that suppression of LCFA uptake by ET‐1 is associated with downregulation of CD36. ET type A receptor (ETAR) antagonist BQ‐610 reversed the IR induction and the ET‐1‐suppressed LCFA uptake. Exogenous replenishment of phosphatidylinositol (PI) 4, 5‐bisphosphate (PIP2) prevented IR induction, but not the suppression of LCFA uptake by ET‐1. Pharmacological inhibition of the activation of mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) completely blocked the ET‐1‐suppressed LCFA uptake. Serving as an inducer of IR, ET‐1 also chronically suppresses LCFA uptake via PIP2‐independent and ERK‐dependent pathway. The interplay between impaired glucose disposal and diminished LCFA utilization, induced by ET‐1, could worsen the dysregulation of adipose metabolism and energy homeostasis in insulin‐resistant states. 相似文献
107.
108.
Srikhanta YN Gorrell RJ Steen JA Gawthorne JA Kwok T Grimmond SM Robins-Browne RM Jennings MP 《PloS one》2011,6(12):e27569
Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis. 相似文献
109.
Scoring schemes of palindrome clusters for more sensitive prediction of replication origins in herpesviruses
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Many empirical studies show that there are unusual clusters of palindromes, closely spaced direct and inverted repeats around the replication origins of herpesviruses. In this paper, we introduce two new scoring schemes to quantify the spatial abundance of palindromes in a genomic sequence. Based on these scoring schemes, a computational method to predict the locations of replication origins is developed. When our predictions are compared with 39 known or annotated replication origins in 19 herpesviruses, close to 80% of the replication origins are located within 2% of the genome length. A list of predicted locations of replication origins in all the known herpesviruses with complete genome sequences is reported. 相似文献
110.
The invertebrate tachykinin-related peptides (TRPs) with the conserved C-terminal sequence FX1GX2Ramide shows sequence similarity to the vertebrate tachykinins after which they are named, and are hypothesized to be ancestrally related. In this study a polyclonal antiserum generated against a locust tachykinin (LomTK I), was used to demonstrate the presence and describe the distribution of LomTK-like immnoreactivity in the CNS and gut of Rhodnius prolixus. Reverse phase high performance liquid chromatography (RP-HPLC) was used in combination with a sensitive radioimmunoassay (RIA) to demonstrate picomolar amounts of immunoreactive material in the CNS, and femptomolar amounts associated with the hindgut. Furthermore, the results from CNS extracts separated by RP-HPLC, suggest that at least two tachykinin isoforms exist in R. prolixus. A hindgut contraction assay was developed to quantify the myotropic effects of selected LomTKs on R. prolixus hindgut contraction. Both LomTK I and II caused an increase in the frequency of hindgut contractions with EC50 values of 3.6x10(-8)M and 3.8x10(-8)M for LomTK I and II, respectively. 相似文献