Single enzyme-based stem-loop and linear primers co-mediated exponential amplification of short gene sequences.

Isothermal DNA amplification only using a Bacillus stearothermophilus (Bst) DNA polymerase such as loop-mediated isothermal amplification typically entails multiple target sites for primer design and is thereby not suited for the amplification of short gene sequences, for example, the sequences with size below 200 nucleotides (nt). Here we present SLIMP, a novel single enzyme-based isothermal amplification of short gene sequence mediated by both stem-loop and linear primers. In SLIMP, a pair of stem-loop primers and a pair of linear primers are specifically designed to recognize only two target sites. Linear primers in SLIMP are similar as conventional PCR primers, but stem-loop primers are the modified linear primers through attaching a stem-loop structure at their 5'-ends. Attributed to this unique primer design, three basic reaction modes including linear-priming, single stem-loop-priming, and double stem-loop-priming amplifications co-mediate the SLIMP process under the function of Bst DNA polymerase. As a proof-of-concept assay, a synthetic 80 nt sequence from hepatitis B virus S gene was used as the template to develop SLIMP. On performance, SLIMP detection possesses high sensitivity and specificity, good selectivity, and the potential for analysing real sample. Therefore, SLIMP is expected as a novel alternative to amplify short gene sequences using a single enzyme.

Association of HindIII-polymorphism in kappa-casein gene with milk, fat and protein yield in holstein cattle.

The aim of this paper was to evaluate the effect of genetic polymorphism of kappa-casein on milk production in Holstein cattle. Two hundred and ten Holstein cows were used in this study. We established genotype structure of cattle population and calculated allelic frequencies based on PCR-RFLP analyses. The three genotypes: AA (69.52%), AB (27.62%), and BB (2.86%) were detected. Frequency of allele A was 83.33%, and of allele B 16.67%. The Holstein cattle kept in Slovak Republic exhibit a high value of homozygosity (0.7222) and low values of polymorphism information content (0.2392), effective number of alleles (1.3847) and level of possible variability realization (27.91%). The effect of polymorphism of CSN3 gene on average breeding values for milk production traits, such as yield of milk, fat and protein expressed in kilograms, as well as percentage content of fat and protein in milk, has been assessed. In our assessment of the observed traits' variability's dependence on CSN3 gene polymorphism, we detected a statistically significant difference between genotypes only in case of the average breeding value for the percentage of protein in milk.

The HindIII and PvuII polymorphisms of lipoprotein lipase (LPL) gene reduce the risk of ischemic stroke (IS): A meta-analysis.

BACKGROUND: Lipoprotein lipase (LPL) polymorphisms were suggested to be the risk factor for ischemic stroke (IS). However, controversial results were obtained. Our objective was to investigate the association of LPL polymorphisms at Ser447Ter, HindIII (+/-), and PvuII (+/-) with IS risk. METHODS: Literatures search were carried out on databases: PubMed, Web of science, the Cochrane database of system reviews, Chinese National Knowledge Infrastructure, and Embase. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated to detect the relationship between LPL polymorphisms and the risk of IS. RESULTS: No significant association was detected between LPL Ser447Ter and IS in allelic, dominant, or recessive models (P > .05). Significant lower frequencies of allelic and dominant models of LPL HindIII (+/-) and PvuII (+/-) in cases were detected (HindIII (+/-): allelic model: P = .0002, OR[95%CI] = 0.80 [0.71, 0.90]; dominant model: P = 0.003, OR[95%CI] = 0.80 [0.69, 0.92]; PvuII (+/-): allelic model: P < 0.0001, OR[95%CI] = 0.75[0.65-0.86]; dominant model: P = 0.02, OR[95%CI] = 0.67[0.48-0.93]). And the recessive model of PvuII (+/-) was significantly associated with the IS risk (P = .01, OR[95%CI] = .71[0.55-0.93]). Subgroup analysis stratified by ethnicity showed that the frequencies of allelic, dominant, and recessive models of HindIII (+/-), as well as dominant model of PvuII (+/-) were significant lower in Asian cases (HindIII (+/-): allelic model: P < .00001, OR[95%CI] = 0.69 [0.59, 0.79]; dominant model: P < .0001, OR[95%CI] = 0.69 [0.58, 0.83]; recessive model: P = .005, OR[95%CI] = 0.66 [0.50, 0.89]; PvuII (+/-): dominant model: P = .0008, OR[95%CI] = 0.66 [0.51-0.84]), but not in Caucasian cases (P > .05). In addition, the frequencies of allelic and recessive models of PvuII (+/-) significantly decreased in Caucasian cases (P < .05). CONCLUSION: the HindIII (+/-) and PvuII (+/-), but not the Ser447Ter might be the protective factors for IS.

Restriction Endonuclease Analysis Typing of Clostridium difficile Isolates.

Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles.

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66 bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy.

Analysis of the HindIII-catalyzed reaction by time-resolved crystallography.

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII-DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

Effects of lipoprotein lipase gene variations, a high-carbohydrate low-fat diet, and gender on serum lipid profiles in healthy Chinese Han youth.

A high-carbohydrate low-fat (HC/LF) diet and lipoprotein lipase gene (LPL) Ser447Stop and Hind III polymorphisms have separately been found to be associated with triacylglycerol (TG) and high density lipoprotein cholesterol (HDL-C). This study sought to test the effects of LPL polymorphisms and an HC/LF diet on the serum lipid profile of Chinese with a lower incidence of coronary artery disease (CAD) consuming a diet with less fat and more carbohydrates. Fifty-six healthy subjects (22.89 ± 1.80 years) were given a control diet of 30.1% fat and 54.1% carbohydrates for 7 days, followed by an HC/LF diet of 13.8% fat and 70.1% carbohydrate for 6 days; there were no changes in the fatty acid composition or restrictions on total energy. Serum lipid profiles at baseline, before and after the HC/LF diet, and LPL polymorphisms were analyzed. After 6 days of the HC/LF diet, TG and the homeostasis model assessment of insulin resistance (HOMAIR) index were found to increase only in females with S447S. No decrease in HDL-C was noted. In subjects with Hind III polymorphism, increased TG was found in all females but not in males. Increased HDL-C, together with apolipoprotein (apo) AI, was found in male H- carriers but not in males with H+/H+ and females. In conclusion, LPL Ser447Stop and Hind III polymorphisms modified the effects of an HC/LF diet on the serum lipid profiles of a young Chinese population in different ways. Effective strategies for dietary interventions targeted at younger populations should take into account the interplay between genetic polymorphisms, diet, and gender.

Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome.

Hepatitis B virus (HBV) pregenomic RNA contains a hairpin structure (ε) located in the preCore region, essential for viral replication. ε stability is enhanced by the presence of preCore variants and ε is recognized by the HBV polymerase (Pol). Mutations in the retrotranscriptase domain (YMDD) of Pol are associated with treatment resistance. The aim of this study was to analyze the preCore region and YMDD motif by ultra-deep pyrosequencing (UDPS). To evaluate the UDPS error rate, an internal control sequence was inserted in the amplicon. A newly developed technique enabled simultaneous analysis of the preCore region and Pol in the same viral genome, as well as the conserved sequence of the internal control. Nucleotide errors in HindIII yielded a UDPS error rate <0.05%. UDPS study confirmed the possibility of simultaneous detection of preCore and YMDD mutations, and demonstrated the complexity of the HBV quasispecies and cooperation between viruses. Thermodynamic stability of the ε signal was found to be the main constraint for selecting main preCore mutations. Analysis of ε-signal variability suggested the essential nature of the ε structural motif and that certain nucleotides may be involved in ε signal functions.

Inhibition of restriction enzymes EcoRI, BamHI and HindIII by phenethylphenylphthalimides derived from thalidomide.

We discovered inhibitors of the restriction enzymes EcoRI, BamHI and HindIII by screening our library of compounds with a phenethylphenylphthalimide skeleton, based on α-glucosidase inhibitors and liver X receptor antagonists derived from thalidomide. Structural development afforded the potent restriction enzyme inhibitors 25 and 26.

DNA exposure to buckminsterfullerene (C60): toward DNA stability, reactivity, and replication.

Buckminsterfullerene (C(60)) has received great research interest due to its extraordinary properties and increasing applications in manufacturing industry and biomedical technology. We recently reported C(60) could enter bacterial cells and bind to DNA molecules. This study was to further determine how the DNA-C(60) binding affected the thermal stability and enzymatic digestion of DNA molecules, and DNA mutations. Nano-C(60) aggregates and water-soluble fullerenols were synthesized and their impact on DNA biochemical and microbial activity was investigated. Our results revealed that water-soluble fullerenols could bind to lambda DNA and improve DNA stability remarkably against thermal degradation at 70-85 °C in a dose-dependent manner. DNase I and HindIII restriction endonuclease activities were inhibited after interacting with fullerenols at a high dose. Experimental results also showed the different influence of fullerenol and nano-C(60) on their antibacterial mechanisms, where fullerenols contributed considerable impact on cell damage and mutation rate. This preliminary study indicated that the application of fullerenols results in significant changes in the physical structures and biochemical functions of DNA molecules.

Decoding circulating nucleic acids in human serum using microfluidic single molecule spectroscopy.

Circulating nucleic acid (CNA) has been the focus of recent research as a noninvasive source of biomarker candidates. Among these markers, DNA fragment size has shown promise for discerning the source of CNA molecules in cancer and prenatal diagnostics. We have developed a one-step assay for analyzing circulating DNA size and quantity directly in human serum. Microfluidic cylindrical illumination confocal spectroscopy and fluorescence burst size analysis are used to individually count and size fluorescently-labeled CNA molecules as they are driven through a microfluidic constriction. First, single molecule sizing was performed on lambda Hind III digest DNA to obtain a size calibration curve. A linear relation between DNA length and burst size was seen from 564 bp to 27.5 kbp. Subsequently, the single molecule assay parameters were optimized. Finally, DNA sizing analysis was performed on serum samples from both early and late stage lung cancer patients. This assay was performed directly in patient serum using only a single reagent, a simple DNA intercalating dye, and without the need for DNA isolation or enzymatic amplification steps. This demonstrates that microfluidic single molecule spectroscopy can be a rapid, facile, and inexpensive alternative to the established PCR-based methods that have been used near exclusively for CNA analysis.

Association of lipase lipoprotein polymorphisms with high-density lipoprotein and triglycerides in elderly men.

Lipoprotein lipase is essential for triglyceride hydrolysis. The polymorphisms S447X in exon 9 and HindIII in intron 8 have been associated with lower triglyceride levels and lower cardiovascular risk in adult men. We examined the association of these lipoprotein lipase polymorphisms with high-density lipoprotein (HDL) and triglyceride levels in elderly men. Blood samples were obtained from 87 elderly men, 48 of whom had cardiovascular disease and 39 (controls) had no history of cardiovascular events. The lipoprotein lipase polymorphisms were analyzed by PCR-RFLP. Allele frequencies were H- = 27.9% and X = 21.5%. There were no significant differences in allele frequencies or blood lipid levels between cardiovascular disease and control groups. However, the X allele was associated with a lower triglyceride/HDL ratio, 2.30 vs 3.02 for X allele absent (P = 0.03); the H-X haplotype was associated with lower triglyceride levels compared to the H+S haplotype (1.22 vs 1.58 mM, respectively) and a lower triglyceride/HDL ratio (2.29 vs 3.26, respectively). The X allele and H-X haplotype were associated with lower triglyceride/HDL ratios in these elderly men, independent of the history of cardiovascular events.

Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations.

The three-dimensional crystal structures of HindIII bound to its cognate DNA with and without divalent cations were solved at 2.17 and 2.00 A resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to the EcoRI-like (alpha-class) subfamily of type II restriction endonucleases. The cognate DNA-complex structures revealed the specific DNA-recognition mechanism of HindIII by which it recognizes the palindromic sequence A/AGCTT. In the Mg(2+) ion-soaked structure the DNA was cleaved and two ions were bound at each active site, corresponding to the two-metal-ion mechanism.

Neuromuscular performance of elite rugby union players and relationships with salivary hormones.

This study compared the neuromuscular performance (speed, power, strength) of elite rugby union players, by position, and examined the relationship between player performance and salivary hormones, by squad and position. Thirty-four professional male rugby players were assessed for running speed (10-m, 20-m or 30-m sprints), concentric mean (MP) and peak power (PP) during a 70-kg squat jump (SJ) and 50-kg bench press throw (BT), and estimated 1 repetition maximum (1RM) strength for a box squat (BS) and bench press (BP). Tests were performed on separate days with absolute and normalized (power and strength only) values computed. Saliva was collected before each test and assayed for testosterone (Sal-T) and cortisol (Sal-C). In absolute terms, the backs demonstrated greater speed and BT MP, whereas the forwards produced greater SJ PP and MP and BS 1RM (p < 0.01). However, BT, SJ and BS performances were no different when normalized for body mass in kg (p > 0.05). A comparison (absolute and normalized) of BT PP showed no positional differences (p > 0.05), whereas BP 1RM was greater for the forwards (p < 0.05). These results may be attributed to genetic and/or training factors relating to the positional demands of rugby. The Sal-T and/or Sal-C concentrations of players correlated to speed, power, and strength, especially for the backs (p < 0.05), thereby confirming relationships between neuromuscular performance and hormone secretion patterns. Based on these findings, it was suggested that training to increase whole-body and muscle mass might facilitate general performance improvements. Training prescription might also benefit from acute and chronic hormone monitoring to identify those individuals likely to respond more to hormonal change.

Molecular epidemiology of Clostridium difficile over the course of 10 years in a tertiary care hospital.

BACKGROUND: The molecular epidemiology of endemic and outbreak Clostridium difficile strains across time is not well known. METHODS: HindIII restriction endonuclease analysis (REA) typing was performed on available clinical C. difficile isolates from 1982 to 1991. RESULTS: The annual incidence of C. difficile infection (CDI) ranged from 3.2 to 9.9 cases per 1000 discharges and was significantly higher in 1982, 1983, 1985, and 1991 (high-incidence years) than in other years (mean standard deviation number of cases for the high- vs the low-incidence years, 121.8 +/-20.4 and 70.0 +/-15.0; P =.002). A total of 696 (76.6%) of 908 C. difficile isolates were available for REA typing over the 10-year period. Large clusters (>or=10 CDI cases in consecutive months) were caused by REA types B1 and B2 in 1982 and 1983, F2 and B1 in 1985, and K1 in 1991 (high-incidence years). Small clusters of 4-9 CDI cases in consecutive months were caused by REA types G1 (1984), Y4 and Y6 (1987), Y2 (1988), L1 (1989), Y1 (1990), and K1 (1991). Current epidemic REA group BI (unrelated to type B1) was isolated 6 times, twice in 1984, 1988, and 1990. CONCLUSIONS: Years with a high incidence of CDI were associated with large clusters of specific REA types that changed yearly. The molecular epidemiology of CDI in this hospital was characterized by a wide diversity of C. difficile types and an ever-changing dominance of specific C. difficile types over time. The current epidemic BI group was found sporadically on 6 occasions. A changing CDI molecular epidemiology should be expected in the future.

Isolation and characterization of bovine herpesvirus 4 (BoHV-4) from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome.

BACKGROUND: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with a Worldwide distribution in cattle and is often isolated from the uterus of animals with postpartum metritis or pelvic inflammatory disease. Virus strain adaptation to an organ, tissue or cell type is an important issue for the pathogenesis of disease. To explore the mechanistic role of viral strain variation for uterine disease, the present study aimed to develop a tool enabling precise genetic discrimination between strains of BoHV-4 and to easily manipulate the viral genome. METHODS: A strain of BoHV-4 was isolated from the uterus of a persistently infected cow and designated BoHV-4-U. The authenticity of the isolate was confirmed by RFLP-PCR and sequencing using the TK and IE2 loci as genetic marker regions for the BoHV-4 genome. The isolated genome was cloned as a Bacterial Artificial Chromosome (BAC) and manipulated through recombineering technology RESULTS: The BoHV-4-U genome was successfully cloned as a BAC, and the stability of the pBAC-BoHV-4-U clone was confirmed over twenty passages, with viral growth similar to the wild type virus. The feasibility of using BoHV-4-U for mutagenesis was demonstrated using the BAC recombineering system. CONCLUSION: The analysis of genome strain variation is a key method for investigating genes associated with disease. A resource for dissection of the interactions between BoHV-4 and host endometrial cells was generated by cloning the genome of BoHV-4 as a BAC.

A HindIII polymorphism of fibronectin gene is associated with nephrolithiasis.

OBJECTIVES: To evaluate the association between fibronectin gene (FN1) polymorphisms and calcium oxalate nephrolithiasis as a genetic risk factor. METHODS: Genomic DNA of 143 patients with calcium oxalate nephrolithiasis and 154 healthy controls were screened for polymorphisms (HaeIII b, MspI, and HindIII) of the FN1 gene, using polymerase chain reaction-restriction fragments length polymorphism method. Allele and genotype frequencies were compared between the groups. RESULTS: Although the observed differences between distribution of genotypes of AA, AB, and BB (for HaeIII b), as well as CC, CD, and DD (MspI) were not significant, FF genotype for HindIII showed significant difference when compared with both EF and EE + EF genotype (P = .00202 and P = .00203, respectively). CONCLUSIONS: The results of our study revealed that HindIII polymorphism of the FN1 gene is highly associated with calcium oxalate stone disease. This association makes FN a good candidate for further studies about the etiology of stone disease, and in the future it could be a candidate marker for evaluating the genetic risks in patients with nephrolithiasis.

Plasminogen activator inhibitor-1 C/G polymorphism in relation to plasma levels in rheumatoid arthritis.

Plasminogen activator inhibitor type 1 (PAI-1) is an inhibitor of plasmin production. Plasmin can directly or indirectly to degrade cartilage and bone matrix. The PAI-1 HindIII polymorphism has been associated with high PAI-1 plasma levels in myocardial infarction patients and control populations. Furthermore, it has been associated with the angiographic extent of coronary artery disease, but their involvement in other diseases is still uncertain. Here, we assessed the relationship between PAI-1 HindIII polymorphism and PAI-1 plasma levels in rheumatoid arthritis (RA). One hundred and twenty-five RA patients and 132 control subjects (CS) were included. Genotypes were identified by the polymerase chain reaction-restriction fragment length polymorphism technique and PAI-1 plasma levels were quantified using an ELISA kit. Not significant differences in genotype and allele frequencies between both studied groups were observed (P > 0.05). RA patients showed lower PAI-1 plasma levels (18.92 +/- 12.94 ng/ml) than CS (23.68 +/- 23.38 ng/ml), without significant difference (P = 0.299). However, in RA patients the C/G genotype carriers showed higher PAI-1 plasma levels (23.00 +/- 13.81 ng/ml) with respect to C/C (16.77 +/- 11.97 ng/ml) and G/G (10.47 +/- 7.07 ng/ml) genotype carriers (P = 0.036). The PAI-1 HindIII polymorphism was not associated with RA susceptibility. However, the C/G genotype is associated with high PAI-1 plasma levels in RA patients.

Permethrin induces lymphocyte DNA lesions at both Endo III and Fpg sites and changes in monocyte respiratory burst in rats.

Pyrethroids are widely used insecticides of low acute toxicity in mammals but the consequences of long-term exposure are of concern. Their insecticidal action is related to neurotoxicity and, in addition, there are indications of mammalian immuno-toxicity. In this work the effect of 60 days permethrin (150 mg kg(-1) body weight/day) exposure on two types of leukocytes (monocytes and lymphocytes) in adolescent rats was investigated. In particular, the monocyte respiratory burst response was first investigated, followed by studies on the degree and type of lymphocyte DNA damage induced by permethrin at this stage of life. Permethrin treatment reduces the monocyte respiratory burst response to phorbol myristate acetate, thereby decreasing superoxide anion (65%) and hydrogen peroxide (37%) production. Moreover an increase [correction made here after initial online publication] in monocyte plasma membrane fluidity in the hydrophilic-hydrophobic interface of the lipid bilayer was measured. Data obtained from the comet assay show that permethrin induces lymphocyte DNA lesions at both formamido pyrimidine glycosylase (Fpg) and endonuclease III (Endo III) sites in adolescent rats. Our results indicate the key role of permethrin in oxidative stress whose consequences lead to biochemical and functional changes. The reduced phagocyte respiratory burst induced by permethrin treatment and the type of DNA damage measured could represent new relevant aspects of pyrethroid toxicity which should be considered for human health.

A HindIII BAC library construction of Mesobuthus martensii Karsch (Scorpiones:Buthidae): an important genetic resource for comparative genomics and phylogenetic analysis.

Scorpions are "living but sophisticated fossils" that have changed little in their morphology since their first appearance over the past 450 million years ago. To provide a genetic resource for understanding the evolution of scorpion genome and the relationships between scorpions and other organisms, we first determined the genome size of the scorpion Mesobuthus martensii Karsch (about 600 Mbp) in the order Scorpiones and constructed a HindIII BAC library of the male scorpion M. martensii Karsch from China. The BAC library consists of a total of 46,080 clones with an average insert size of 100 kb, providing a 7.7-fold coverage of the scorpion haploid genome size of 600 Mbp as revealed in this study. High-density colony hybridization-based library screening was performed using 18S-5.8S-28S rRNA gene that is one of the most commonly used phylogenetic markers. Both library screening and PCR identification results revealed six positive BAC clones which were overlapped, and formed a contig of approximately 120 kb covering the rDNA. BAC DNA sequencing analysis determined the complete sequence of M. martensii Karsch rDNA unit that has a total length of 8779 bp, including 1813 bp 18s rDNA, 157 bp 5.8s rDNA, 3823 bp 28s rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2. Interestingly, some tandem repeats are present in the rRNA intergenic sequence (IGS) and ITS1/2 regions. These results demonstrated that the BAC library of the scorpion M. martensii Karsch and the complete sequence of rDNA unit will provide important genetic resources and tools for comparative genomics and phylogenetic analysis.