Recovery of Small DNA Fragments from Serum Using Compaction Precipitation

Background

While most nucleic acids are intracellular, trace amounts of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including micro RNAs, can also be found in peripheral blood. Many studies have suggested the potential utility of these circulating nucleic acids in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. However, DNA circulating in blood is usually present at very low concentrations (ng/ml), and is in the form of relatively small fragments (<1,000 bp), making its isolation challenging.

Methods

Here we report an improved method for the isolation of small DNA fragments from serum using selective precipitation by quaternary ammonium compaction agents. A 151 bp fragment of double-stranded DNA from the Escherichia coli bacteriophage lambda served as the model DNA in our experiments. DNA was serially diluted in serum until undetectable by conventional polymerase chain reaction (PCR), before being enriched by compaction precipitation.

Results

Starting with concentrations two to three orders of magnitude lower than the PCR-detectable level (0.01 ng/ml), we were able to enrich the DNA to a detectable level using a novel compaction precipitation protocol. The isolated DNA product after compaction precipitation was largely free of serum contaminants and was suitable for downstream applications.

Conclusions

Using compaction precipitation, we were able to isolate and concentrate small DNA from serum, and increase the sensitivity of detection by more than four orders of magnitude. We were able to recover and detect very low levels (0.01 ng/ml) of a small DNA fragment in serum. In addition to being very sensitive, the method is fast, simple, inexpensive, and avoids the use of toxic chemicals.     

Uncontrolled admixture and loss of genetic diversity in a local Vietnamese pig breed

The expansion of intensive livestock production systems in developing countries has increased the introduction of highly productive exotic breeds facilitating indiscriminate crossbreeding with local breeds. In this study, we set out to investigate the genetic status of the Vietnamese Black H'mong pig breed by evaluating (1) genetic diversity and (2) introgression from exotic breeds. Two exotic breeds, namely Landrace and Yorkshire used for crossbreeding, and the H'mong pig population from Ha Giang (HG) province were investigated using microsatellite markers. Within the province, three phenotypes were observed: a White, a Spotted and a Black phenotype. Genetic differentiation between phenotypes was low (0.5-6.1%). The White phenotypes showed intermediate admixture values between exotic breeds and the Black HG population (0.53), indicating a crossbreed status. Management practices were used to predict the rate of private diversity loss due to exotic gene introgressions. After 60 generations, 100% of Black private alleles will be lost. This loss is accelerated if the admixture rate is increased but can be slowed down if the mortality rate (e.g., recruitment rate) is decreased. Our study showed that a large number of markers are needed for accurately identifying hybrid classes for closely related populations. While our estimate of admixture still seems underestimated, genetic erosion can occur very fast even through indiscriminate crossbreeding.     

Anticoccidial Activity of Berberine against Eimeria-Infected Chickens

Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.     

Human Taeniasis and Cysticercosis and Related Factors in Phu Tho Province,Northern Vietnam

Several factors presumed to facilitate the transmission of Taenia spp. were reported in Vietnam. We conducted a cross-sectional study taking questionnaires from 1,185 participants, and collecting 1,151 sera and 1,036 stool samples in northern Vietnam. Sera were examined for circulating antigens of Taenia solium cysticerci using ELISA, stools for Taenia eggs by Kato-Katz smear, and copro-antigens by ELISA. Ag-ELISA revealed 4.6% antigen positivity, indicating infection with viable cysticerci. Taenia eggs were detected in 1.5% of participants. Copro-antigens were found in 2.8% of participants. Eating raw meat and/or vegetables was significantly associated with the presence of copro-antigen (OR=8.6, 95% CI: 1.16–63.9, P=0.01). Considering the high taeniasis prevalence and the associated threat, public health attention should be given to treat the tapeworm carriers in the projected areas.     

CT-based assessment of regional pulmonary microvascular blood flow parameters.

To determine regional pulmonary microvascular mean transit times (MTTs), we used electrocardiogram-gated X-ray computed tomographic imaging to follow bolus radiopaque contrast material through the lungs in anesthetized animals (7 dogs and 1 pig, prone and supine). By deconvolution/reconvolution of regional time-attenuation curves obtained from parenchyma and large lobar arteries, we estimated the microvascular residue function and reconstituted the regional microvascular time-attenuation curves and, thus, regional microvascular MTTs. The mean microvascular MTTs in the supine and prone postures were 3.94 +/- 1.0 and 3.40 +/- 0.84 (mean +/- SD), respectively. The dependent-nondependent vertical gradient of MTT was greater in the supine [slope = 0.25 +/- 0.10 (SD), P < 0.001 by t-test] than in the prone (-0.03 +/- 0.06 in 6 of 8 animals; 2 outliers had positive slopes) posture. In both postures, there was a trend toward faster transit times in the dorsal-basal lung region in six of the eight animals, suggesting gravity-independent higher vascular conductance dorsocaudally. We conclude that deconvolution methods, in association with electrocardiogram-gated high-speed X-ray computed tomography, can provide insights into regional heterogeneity of pulmonary microvascular MTT in vivo.     

Computing phylogenetic diversity for split systems

In conservation biology it is a central problem to measure, predict, and preserve biodiversity as species face extinction. In 1992 Faith proposed measuring the diversity of a collection of species in terms of their relationships on a phylogenetic tree, and to use this information to identify collections of species with high diversity. Here we are interested in some variants of the resulting optimization problem that arise when considering species whose evolution is better represented by a network rather than a tree. More specifically, we consider the problem of computing phylogenetic diversity relative to a split system on a collection of species of size n. We show that for general split systems this problem is NP-hard. In addition we provide some efficient algorithms for some special classes of split systems, in particular presenting an optimal O(n) time algorithm for phylogenetic trees and an O(n log n + nk) time algorithm for choosing an optimal subset of size k relative to a circular split system.     

Identification of parasporin genes in Vietnamese isolates of Bacillus thuringiensis

Four genes encoding parasporins, cytotoxins preferentially killing human cancer cells in vitro, were isolated from four Vietnamese strains of Bacillus thuringiensis. Nucleotide sequence analysis revealed that: (1) three genes fall into the two known classes, ps1Aa and ps1Ab, and (2) another one belongs to ps1Ac, a novel gene class established in this study. Upon proteolytic activation, parasporal protein of the organism with ps1Ac exhibited strong cytocidal activity against human cancer cells, HeLa and Hep G2, but not to non-cancer normal cells, UtSMC and HC.     

NMR investigation of chitosan derivatives formed by the reaction of chitosan with levulinic acid

Chitosan derivatives are obtained by reaction of chitosan with a low degree of acetylation and levulinic acid under different experimental conditions. The chemical structure of the different derivatives obtained is determined using ¹H and ¹³C NMR spectroscopies. The intrinsic viscosity is used to follow the molecular weight evolution. Finally, conditions are described in which water-soluble N-carboxybutylchitosan is obtained. In particular, the time of the reduction step and the ratio between reagents are investigated. Under mild conditions and short times of reduction there is a very low degree of substitution and only the monocarboxybutylchitosan is formed. The dicarboxylated form is never observed. The cyclic derivative (5-methylpyrrolidinone chitosan) is obtained when the reducing agent is added slowly to the reactants.     

Thymidylate synthase: a novel genetic determinant of plasma homocysteine and folate levels

The thymidylate synthase gene ( TYMS or TS) encodes a tightly regulated enzyme that catalyzes the conversion of deoxyuridylate to thymidylate, and contains a tandem repeat polymorphism that affects expression of the enzyme. We have investigated the relationship between TYMS genotype and plasma concentrations of homocysteine and folate in a cohort of 505 Chinese from Singapore. TYMS 3/3 genotype was associated with reduced plasma folate and, among individuals with low dietary folate intake, with elevated plasma homocysteine levels. These associations were independent of the well-established methylenetetrahydrofolate reductase ( MTHFR) C677T genotype effects on plasma folate and homocysteine levels. Our results suggest that TYMS and MTHFR compete for limiting supplies of folate required for the remethylation of homocysteine. These genetic determinants of plasma folate and homocysteine levels may be useful in identifying individuals at increased risk for cardiovascular disease.