RNA secondary structure formation: a solvable model of heteropolymer folding

The statistical mechanics of heteropolymer structure formation is studied in the context of RNA secondary structures. A designed RNA sequence biased energetically towards a particular native structure (a hairpin) is used to study the transition between the native and molten phase of the RNA as a function of temperature. The transition is driven by a competition between the energy gained from the polymer's overlap with the native structure and the entropic gain of forming random contacts. A simplified Go-like model is proposed and solved exactly. The predicted critical behavior is verified via exact numerical enumeration of a large ensemble of similarly designed sequences.Comment: 4 pages including 2 figure

Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism

<p>Abstract</p> <p>Background</p> <p>Many SNP discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. However, SNPs are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. In this study, a simple, sequence-adaptable SNP discrimination mechanism between a 'wild-type' and 'mutant' template is demonstrated. This model differs from other artificial restriction endonuclease models as <it>cis- </it>rather than <it>trans-</it>orientated regions of single stranded DNA were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted.</p> <p>Results</p> <p>A series of mismatch 'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to S1 nuclease. In this model, generation of oligonucleotide-mediated ssDNA mismatch 'bubbles' in the presence of S1 nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. Increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. This level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in Tm (~5.0°C per base-pair mismatch) and to optimise annealing conditions (~18.38°C below Tm) of the mismatched oligonucleotide sets.</p> <p>Conclusions</p> <p>The <it>in vitro </it>'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more SNPs. Furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine Tm and annealing rates as an alternative to conventional hybridisation detection strategies.</p

Lead and δ-Aminolevulinic Acid Dehydratase Polymorphism: Where Does It Lead? A Meta-Analysis

BACKGROUND: Lead poisoning affects many organs in the body. Lead inhibits δ-aminolevulinic acid dehydratase (ALAD), an enzyme with two co-dominantly expressed alleles, ALAD1 and ALAD2. OBJECTIVE: Our meta-analysis studied the effects of the ALAD polymorphism on a) blood and bone lead levels and b) indicators of target organ toxicity. DATA SOURCE: We included studies reporting one or more of the following by individuals with genotypes ALAD1-1 and ALAD1-2/2-2: blood lead level (BLL), tibia or trabecular lead level, zinc protoporphyrin (ZPP), hemoglobin, serum creatinine, blood urea nitrogen (BUN), dimercaptosuccinic acid–chelatable lead, or blood pressure. DATA EXTRACTION: Sample sizes, means, and standard deviations were extracted for the genotype groups. DATA SYNTHESIS: There was a statistically significant association between ALAD2 carriers and higher BLL in lead-exposed workers (weighted mean differences of 1.93 μg/dL). There was no association with ALAD carrier status among environmentally exposed adults with BLLs < 10 μg/dL. ALAD2 carriers were potentially protected against adverse hemapoietic effects (ZPP and hemoglobin levels), perhaps because of decreased lead bioavailability to heme pathway enzymes. CONCLUSION: Carriers of the ALAD2 allele had higher BLLs than those who were ALAD1 homozygous and higher hemoglobin and lower ZPP, and the latter seems to be inversely related to BLL. Effects on other organs were not well delineated, partly because of the small number of subjects studied and potential modifications caused by other proteins in target tissues or by other polymorphic genes

High intakes of choline and betaine reduce breast cancer mortality in a population-based study

Choline and betaine provide methyl groups for one-carbon metabolism. Humans obtain these nutrients from a wide range of foods. Betaine can also be synthesized endogenously from its precursor, choline. Although animal studies have implied a causal relationship between choline deficiency and carcinogenesis, the role of these two nutrients in human carcinogenesis and tumor progression is not well understood. We investigated the associations of dietary intakes of choline and betaine and breast cancer risk and mortality in the population-based Long Island Breast Cancer Study Project. Among the 1508 case-group women, 308 (20.2%) deaths occurred, among whom 164 (53.2%) died of breast cancer by December 31, 2005. There was an indication that a higher intake of free choline was associated with reduced risk of breast cancer (P trend=0.04). Higher intakes of betaine, phosphocholine, and free choline were associated with reduced all-cause as well as breast cancerspecific mortality in a dose-dependent fashion. We also explored associations of polymorphisms of three key choline- and betaine-metabolizing genes and breast cancer mortality. The betaine-homocysteine methyltransferase gene (BHMT) rs3733890 polymorphism was associated with reduced breast cancer-specific mortality (hazard ratio, 0.64; 95% confidence interval, 0.42-0.97). Our study supports the important roles of choline and betaine in breast carcinogenesis. It suggests that high intake of these nutrients may be a promising strategy to prevent the development of breast cancer and to reduce its mortality

Comprehensive Analysis of 5-Aminolevulinic Acid Dehydrogenase (ALAD) Variants and Renal Cell Carcinoma Risk among Individuals Exposed to Lead

BACKGROUND: Epidemiologic studies are reporting associations between lead exposure and human cancers. A polymorphism in the 5-aminolevulinic acid dehydratase (ALAD) gene affects lead toxicokinetics and may modify the adverse effects of lead. METHODS: The objective of this study was to evaluate single-nucleotide polymorphisms (SNPs) tagging the ALAD region among renal cancer cases and controls to determine whether genetic variation alters the relationship between lead and renal cancer. Occupational exposure to lead and risk of cancer was examined in a case-control study of renal cell carcinoma (RCC). Comprehensive analysis of variation across the ALAD gene was assessed using a tagging SNP approach among 987 cases and 1298 controls. Occupational lead exposure was estimated using questionnaire-based exposure assessment and expert review. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression. RESULTS: The adjusted risk associated with the ALAD variant rs8177796(CT/TT) was increased (OR = 1.35, 95%CI = 1.05-1.73, p-value = 0.02) when compared to the major allele, regardless of lead exposure. Joint effects of lead and ALAD rs2761016 suggest an increased RCC risk for the homozygous wild-type and heterozygous alleles ((GG)OR = 2.68, 95%CI = 1.17-6.12, p = 0.01; (GA)OR = 1.79, 95%CI = 1.06-3.04 with an interaction approaching significance (p(int) = 0.06). No significant modification in RCC risk was observed for the functional variant rs1800435(K68N). Haplotype analysis identified a region associated with risk supporting tagging SNP results. CONCLUSION: A common genetic variation in ALAD may alter the risk of RCC overall, and among individuals occupationally exposed to lead. Further work in larger exposed populations is warranted to determine if ALAD modifies RCC risk associated with lead exposure

Role of Cell-to-Cell Variability in Activating a Positive Feedback Antiviral Response in Human Dendritic Cells

In the first few hours following Newcastle disease viral infection of human monocyte-derived dendritic cells, the induction of IFNB1 is extremely low and the secreted type I interferon response is below the limits of ELISA assay. However, many interferon-induced genes are activated at this time, for example DDX58 (RIGI), which in response to viral RNA induces IFNB1. We investigated whether the early induction of IFNBI in only a small percentage of infected cells leads to low level IFN secretion that then induces IFN-responsive genes in all cells. We developed an agent-based mathematical model to explore the IFNBI and DDX58 temporal dynamics. Simulations showed that a small number of early responder cells provide a mechanism for efficient and controlled activation of the DDX58-IFNBI positive feedback loop. The model predicted distributions of single cell responses that were confirmed by single cell mRNA measurements. The results suggest that large cell-to-cell variation plays an important role in the early innate immune response, and that the variability is essential for the efficient activation of the IFNB1 based feedback loop

Massively Parallel Haplotyping on Microscopic Beads for the High-Throughput Phase Analysis of Single Molecules

In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of less than 1∶10,000. We also accurately quantified chimera formation during the exponential phase of PCR by different DNA polymerases