A simple technology for plastid transformation with fragmented DNA.

Plastid engineering has several unique advantages such as high expression of transgenes due to high polyploidy of plastid genomes and environmental biosafety because of maternal inheritance of transgenes, and has become a promising tool for molecular farming, metabolic engineering, and genetic improvement. However, there are no standard vectors available for plastid transformation. Moreover, the construction of plastid transformation vectors containing long operons or genes encoding proteins that are toxic to Escherichia coli was tedious or difficult. Here, we developed a simple plastid transformation technology without the need for in vitro vector construction by using multiple linear DNA fragments which share homologous sequences (HSs) at their ends. The strategy is based on homologous recombination between HSs of DNA fragments via endogenous recombination machinery in plastids, which subsequently are integrated into the plastid genome. We found that HSs of 200 bp or longer were sufficient for mediating the integration into the plastid genome with at least similar efficiency to that of plasmid DNA-based plastid transformation. Furthermore, we successfully used this method to introduce a phage lysin-encoding gene and a long operon into a tobacco plastid genome. The establishment of this technology simplifies the plastid transformation procedure and provides a novel solution for expressing proteins, which are either toxic to the cloning host or large operons in plastids, without need of vector cloning.

Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems.

Biological samples, including skeletal remains exposed to environmental insults for extended periods of time, exhibit increasing levels of DNA damage and fragmentation. Human forensic identification methods typically use a combination of mitochondrial (mt) DNA sequencing and short tandem repeat (STR) analysis, which target segments of DNA ranging from 80 to 500 base pairs (bps). Larger templates are often unavailable as skeletal samples age and the associated DNA degrades. Single-nucleotide polymorphism (SNP) loci target shorter templates and may serve as a solution to the problem. Recently developed assays for STR and SNP analysis using a massively parallel sequencing approach, such as the ForenSeq kit (Verogen, San Diego, CA), offer a means for generating results from degraded samples as they target templates down to 60 to 170 bps. We performed a modeling study that demonstrates that SNPs can increase the significance of an identification when analyzing DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. Observations from this study were then compared with human skeletal material results (n = 14, ninth to eighteenth centuries), which further demonstrated the utility of the ForenSeq kit for degraded samples. The robustness of the Promega PowerSeq™ Mito System was also tested with human skeletal remains (n = 70, ninth to eighteenth centuries), resulting in successful coverage of 99.29% of the mtDNA control region at 50× coverage or more. This was accompanied by modifications to a mainstream DNA extraction technique for skeletal remains that improved recovery of shorter templates.

Influence of oxidative stress on the antibacterial activity of betulin, betulinic acid and ursolic acid.

Contribution of reactive oxygen species and oxidative stress in the antibacterial activities of betulin, betulinic acid and ursolic acid against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus was investigated. The minimum inhibitory concentrations of betulin, betulinic acid and ursolic acid against E. coli, P. aeruginosa and S. aureus are 1024-, 256- and 1024-µg/mL; 512-, 256- and 256 µg/mL; 256-, 256- and 64 µg/mL respectively. Cell viability of betulin-, betulinic acid- and ursolic acid-treated bacteria decrease in time dependent manner. Treatment of bacteria in the presence of 2,2'-bipyrydyl increased cell viability. Superoxide anion radical production increased significantly (p < 0.05) in bacterial cells-treated with betulin, betulinic acid and ursolic acid. Furthermore, NAD+/NADH ratio increased significantly (p < 0.05) in betulin-, betulinic acid- and ursolic acid-treated bacteria. Similarly, level of reduced glutathione in E. coli, P. aeruginosa and S. aureus decreased significantly with corresponding increase in glutathione disulphide, malondialdehyde and fragmented DNA following betulin, betulinic acid and ursolic acid treatments. It is evident from the above findings that betulin, betulinic acid and ursolic acid enhanced electron transport chain activity in E. coli, P. aeruginosa and S. aureus leading to increased ROS generation, Fenton reaction, lipid peroxidation, fragmented DNA and consequentially bacterial death.

Hepatoprotective potential of Phyllanthus muellarianus leaf extract: studies on hepatic, oxidative stress and inflammatory biomarkers.

CONTEXT: Leaves of Phyllanthus muellarianus (Kuntze) Exell. (Euphorbiacea) are widely used in the management of liver disorders in Nigeria. However, no there is no scientific validation to support this use. OBJECTIVE: Hepatoprotective effect of Phyllanthus muellarianus aqueous leaf extract was investigated in acetaminophen-induced liver injury mice. MATERIALS AND METHODS: Hepatoprotective effect of Phyllanthus muellarianus aqueous leaf extract was evaluated in acetaminophen-induced hepatic damage in Swiss albino mice using biomarkers of hepatocellular indices, oxidative stress, proinflammatory factors and lipid peroxidation. Mice received distilled water, 100, 200, or 400 mg/kg b.w of Phyllanthus muellarianus aqueous leaf extract, respectively, for seven days. Treatment groups were challenged with 300 mg/kg b.w of acetaminophen on the sixth day. RESULTS: Oral administration of Phyllanthus muellarianus aqueous leaf extract significantly (p < 0.05) attenuates acetaminophen-mediated alterations in serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin and total bilirubin by 76.56, 85.41, 89.39, 82.77 and 78.38%. Similarly, acetaminophen-mediated decrease in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase were significantly attenuated in the liver of mice by 85.10, 80.81, 80.45, 76.23 and 95.22%, respectively. Increased levels of conjugated dienes, lipid hydroperoxides, malondialdehyde, protein carbonyl, fragmented DNA, tumor necrosis factor-α, interleukin-6 and -8 were significantly lowered by Phyllanthus muellarianus aqueous leaf extract. CONCLUSION: Overall, results of this study show that Phyllanthus muellarianus halted acetaminophen-mediated hepatotoxicity due to its capability to enhance antioxidant enzymes.

Differential pre-amplification of STR loci for fragmented forensic DNA profiling.

DNA profiling of short tandem repeats (STR) has been successfully used for the identification of individuals in forensic samples, accidents and natural disasters. However, STR profiling of DNA isolated from old crime scenes and damaged biological samples is difficult due to DNA degradation and fragmentation. Here, we show that pre-amplification of STR loci using biotinylated primers for the STR loci is an efficient strategy to obtain STR profiling results from fragmented forensic samples. Analysis of STR loci with longer amplicon sizes is generally hampered, since these relatively long loci are vulnerable to DNA fragmentation. This problem was overcome by using reduced or increased primer concentrations for loci with shorter or longer amplicon sizes, respectively, in our pre-amplification strategy. In addition, pre-amplification of STR loci into two groups of short or long amplicon size increases the efficiency of STR profiling from highly fragmented forensic DNA samples. Therefore, differential pre-amplification of STR loci is an effective way to obtain DNA profiling results from fragmented forensic samples.

Redox and respiratory chain related alterations in the lophirones B and C-mediated bacterial lethality.

The influence of chalcone dimers, lophirones B and C on redox status and respiratory chain activity of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa was investigated. Minimum inhibitory concentrations (MIC) of lophirones B and C against E. coli, P. aeruginosa and S. aureus are 200-; 100-; 200- and 150-µg/mL respectively. Similarly, the minimum bactericidal concentrations (MBC) of lophirones B and C are 250; 200; 300 and 200-µg/mL respectively. The optical densities and colony forming units of lophirones B and C-treated bacteria decreased in time-dependent manner. Superoxide anion content of E. coli, P. aeruginosa and S. aureus exposed to lophirones B and C (4× MIC) increased significantly. Superoxide dismutase and catalase in the chalcone dimers-treated bacteria increased significantly. Conversely, reduced glutathione in lophirones B and C-treated bacteria decrease significantly with corresponding increase in glutathione disulfide. Furthermore, malondialdehyde and fragmented DNA increased significantly following exposure to the chalcone dimers. The respiratory complex I and II decreased significantly in the chalcone dimers-treated bacteria. From the findings, lophirones B and C altered intracellular redox status via enhanced oxidant generation possibly by autoxidation, Fenton chemistry and inhibiting electron transport chain resulting to lipid peroxidation and DNA fragmentation and consequentially bacterial cell death.

Risk to fragmented DNA in dry, wet, and frozen states from computed tomography: a comparative theoretical study.

Computed tomography represents the gold standard in forensic and palaeopathological diagnosis. However, the X-rays used may affect the DNA quality through fragmentation and loss of genetic information. Previous work showed that the effects of ionizing radiation on dry DNA are non-significant with P < 10(-8), which cannot be detected by means of polymerase chain reaction methods. In the present paper, complete analytical model that characterizes radiation effects on fragmented DNA in dry, wet, and frozen states is described. Simulation of radiation tracks in water phantom cells was performed using the Geant4-DNA toolkit. Cell hits by electrons with energies between 5 and 20 keV were simulated, and the formation of radiolytic products was assessed at a temperature of 298 K. The diffusion coefficient and the mean square displacement of reactive species were calculated by Stokes-Einstein-Smoluchowski relations at 273 K. Finally, DNA fragment damage was estimated using the density distribution of fragments calculated from atomic force microscopy images. The lowest probability of radiation-induced DNA damage was observed for dry state, with a range from 2.5 × 10(-9) to 7.8 × 10(-12) at 298 K, followed by that for frozen state, with a range from 0.9 to 4 × 10(-7) at 273 K. The highest probability of radiation-induced DNA damage was demonstrated for fragmented DNA in wet state with a range from 2 to 9 × 10(-7) at 298 K. These results significantly improve the interpretation of CT imaging in future studies in forensic and palaeopathological science.

Lophirones B and C prevent aflatoxin B1-induced oxidative stress and DNA fragmentation in rat hepatocytes.

Context Despite the reported anticarcinogenic activity of lophirones B and C, no scientific information exists for its activity in rat hepatocytes. Objective Effect of lophirones B and C on aflatoxin B1 (AFB1)-induced oxidative stress, and DNA fragmentation in rat hepatocytes was investigated. Materials and methods Wistar rat hepatocytes were incubated with lophirones B and C (1 mg/mL) or sylimarin (1 mg/mL) in the presence or absence of AFB1. For an in vivo study, rats were orally administered with lophirones B and C, and/or AFB1 (20 µg/d) for 9 weeks. Results Lophirones B and C lowered AFB1-mediated increase in nitric oxide, superoxide anion radicals, caspase-3 and fragmented DNA. Lophirones B and C attenuated AFB1-mediated decrease in superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and reduced glutathione. Also, lophirones B and C attenuated AFB1-mediated increase in conjugated dienes, lipid hydroperoxides and malondialdehyde in rat hepatocytes. Furthermore, AFB1-mediated alterations in alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin, total bilirubin and globulin in rat serum were significantly annulled in lophirones B and C-treated rats. Conclusion This study revealed that lophirones B and C prevented AFB1-induced oxidative damage in rat hepatocytes.

Corneocyte lysis and fragmented DNA considerations for the cellular component of forensic touch DNA.

DNA deposited by individuals' hands is a routine part of forensic analysis, yet little is understood about the precise cellular contents left by handling. "Dead" skin cells known as corneocytes make up the majority of the cellular material left in touch deposits by people's hands but are known to lack nuclei, making their DNA content ambiguous. Here we measure DNA released from anucleate corneocytes following various lysis methods to determine how much DNA may be present in these cells and how best to recover it from inside the cornified envelope. We demonstrate that enhanced lysis methods using a reducing agent and longer incubation may be valuable for hand deposit samples. Corneocyte DNA can be characterized as highly degraded based on the quantification, STR profiling and fluorescence microscopy of the cells from freshly washed hands. Purification to target shorter DNA fragments is demonstrated. DNA from the washed corneocyte cells is shown to constitute the majority of recoverable DNA with these methods. We consider the use of new methods adapted to cornified cells and fragmented DNA for future research into this sample type.

Exploration of cell-free DNA (cfDNA) recovery for touch deposits.

Although touch deposit DNA is widely used in forensic casework, its cellular and acellular contents and their biological origins are poorly understood. There is evidence that the cell-free component of DNA deposited by handling may contribute substantial genetic information; however, most research into touch DNA recovery does not separate cellular and cell-free fractions or seek to characterize their contents. This work is an important early step in developing methods to isolate the cfDNA from biological material deposited by handling. Size-filtration as a separation technique was determined to be prone to DNA loss, even on optimized control samples of pure ladder DNA. Centrifugal separation was optimized to determine minimum speed and time required to reliably remove all cellular debris from the material collected by rinsing donor hands. To determine if the centrifugal force risked rupturing shed corneocyte cells and releasing cellular DNA into the supernatant, DNA levels were measured, and cells were visualized microscopically before and after centrifugation of hand rinses. Heated buccal cells were used as a positive control to demonstrate cell rupture would be detected with these methods. Following the determination of a suitable separation technique, an investigation into purification methods for cfDNA was conducted. DNA recovery using three kits for plasma cfDNA, one for PCR clean-up and one for genomic DNA were assessed on both ladder DNA to simulate cfDNA fragments and on collected hand deposit supernatants from both unwashed and washed hands. Purification methods designed for recovery of short DNA fragments from plasma yielded the highest recovery percentage across sample types, with BioChain cfPure performing the best. Donors' hands were shown to shed high levels of cfDNA, which were better recovered with a method for short fragments than with a traditional genomic technique often used on touch DNA samples.

Therapeutic Effects of Nano-HAp in a Rat Model of AlCl3 Induced Neurotoxicity.

With the advance in nanomedicine, the present study was conducted to explore the possible therapeutic role of intravenous nano- hydroxyapatite (nano-HAp) in male rats after chronic exposure to aluminum chloride (AlCl3). This exposure interposed DNA fragmentation, apoptosis, alters oxidant/antioxidant status as well as change in content of neurotransmitters. The rats were injected with 100 mg/kg. body weight (b.w.) of AlCl3 intraperitoneally for 90 days, after then nano-HAp was injected intravenously (i.v.) three times per week at a dose level 100 mg/kg b.w. Based on the results obtained, it can be concluded that the treatment with the prepared nano-HAp restrains the damage inflicted on brain modulation by lipid oxidation products and decreased the susceptibility of apoptotic cells death with subsequent repaired the fragmented DNA as well as improved the synthesis of neurotransmitters. The most salient finding of nano-HAp treatment is the disappearance of most pathological changes due to AlCl3 administration.

Contributions of RecA and RecBCD DNA repair pathways to the oxidative stress response and sensitivity of Acinetobacter baumannii to antibiotics.

OBJECTIVES: RecA and RecBCD are responsible for the repair of oxidative DNA damage in bacteria, including Acinetobacter baumannii (A. baumannii). This study evaluated the contribution of recA, recB, recC and recD to the sensitivity and oxidative response of A. baumannii to antibiotics. RESULTS: Inactivation of recA, recB, recC and recD significantly increased the susceptibility of A. baumannii AB5075 to colistin, gentamicin, rifampicin and tigecycline. Furthermore, superoxide anion radicals (•O2-) and hydrogen peroxide (H2O2) accumulated in colistin, gentamicin, rifampicin or tigecycline-treated ΔrecA, ΔrecB, ΔrecC and ΔrecD mutants compared with the parental strain. Concomitantly, a more pronounced increase in fragmented DNA was observed in the mutants compared with the parental strain upon antibiotic treatment. Chelation of ferrous ion (Fe2+) with dipyridyl lowered the susceptibility of ΔrecA, ΔrecB, ΔrecC and ΔrecD strains of A. baumannii to colistin, gentamicin and rifampicin, but not tigecycline, to a level comparable with the parental strain. Antibiotic-mediated accumulation of reactive oxygen species depleted glutathione, with a more profound response in the mutants compared with the parental strain. The antibiotics, except tigecycline, raised the oxidized nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide and adenosine diphosphate/adenosine triphosphate ratio of ΔrecA, ΔrecB, ΔrecC and ΔrecD mutants compared with the parental strain. CONCLUSION: Reduced capability of ΔrecA, ΔrecB, ΔrecC and ΔrecD mutants to repair DNA raised the susceptibility of A. baumannii to colistin, gentamicin, rifampicin and tigecycline. The available data further support the notion that oxidative stress contributes to antibiotic-mediated bacterial killing.

Involvement of oxidative stress in protocatechuic acid-mediated bacterial lethality.

The involvement of oxidative stress in protocatechuic acid-mediated bacterial lethality was investigated. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) of protocatechuic acid against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus are 600 and 700 µg/ml, 600 and 800 µg/ml, and 600 and 800 µg/ml, respectively. The optical densities and colony-forming units of protocatechuic acid-treated bacteria decreased in time-dependent manner. Protocatechuic acid (4× MIC) significantly increased the superoxide anion content of E. coli, P. aeruginosa, and S. aureus compared to dimethyl sulfoxide (DMSO). Superoxide dismutase, catalase, and NAD+ /NADH in protocatechuic acid-treated E. coli, P. aeruginosa, and S. aureus increased significantly when compared to DMSO. Conversely, level of reduced glutathione decreased in protocatechuic acid-treated E. coli, P. aeruginosa, and S. aureus, while glutathione disulfide increased when compared to DMSO. Furthermore, malondialdehyde and fragmented DNA increased significantly following exposure to protocatechuic acid. Protocatechuic acid inhibited the activity of complexes I and II. From the above findings, protocatechuic acid enhanced the generation of reactive oxygen species (superoxide anion radical and hydroxyl radical) in E. coli, P. aeruginosa, and S. aureus, possibly by autoxidation, fenton chemistry, and inhibiting electron transport chain resulting in lipid peroxidation and DNA fragmentation and consequentially bacterial cell death.

Nucleotide carriers for anti-tumour actinomycin antibiotics.

We have investigated a number of complexes of 7-aminoactinomycin D (7AAMD), with its potential carriers: caffeine, folic acid (FA), purine bases-guanine and adenine, pyrimidine base-thymine and with fragmented DNA to determine more stable and suitable complex. The process of binding of the fluorescent antibiotic with clusters of caffeine, guanine, adenine, thymine and with fragmented DNA was accompanied by a considerable long-wavelength shift in excitation spectrum. The energy of interaction between phenoxazine hetero-cycle of 7AAMD and chromophores of the carriers studied has been found. In the case of 7AAMD with guanine, adenine, thymine and caffeine, the energy is about of 7 kcal/mol, which is a little lower than in the case with DNA (7.7 kcal/mol). On the basis of emission spectra, in all examined compounds, with the exception DNA, the 7AAMD molecule emits photons from water phase, not from a cluster, since photo-excitation leads to desorption of the antibiotic from a cluster surface. We observed also the mutual fluorescence quenching of 7AAMD and FA in their complex. It may well be that this complex forms due to interaction of peptide-lactone rings of 7AAMD with system of FA. In the case of DNA, the complex with 7AAMD has very high stability that is determined not only by interaction between phenoxazine of 7AAMD and the DNA bases, but it is largely owing to the interaction between two peptide-lactone rings of 7AAMD and the DNA deoxyribose-phosphate chains.

Contribution of reactive oxygen species to (+)-catechin-mediated bacterial lethality.

The contribution of reactive oxygen species to (+)-catechin-mediated bacterial lethality was investigated. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) of (+)-catechin against E. coli, P. aeruginosa and S. aureus were investigated using 96-well microtitre plate. MIC and MBC of (+)-catechin against E. coli, P. aeruginosa and S. aureus are 600 and 700; 600 and 800; 600 and 800 µg/mL respectively. The optical densities and colony forming units of (+)-catechin-treated bacteria decreased. (+)-Catechin (4× MIC) significantly increased the superoxide anion content of E. coli, P. aeruginosa and S. aureus compared to DMSO. Superoxide dismutase and catalase in (+)-catechin treated E. coli, P. aeruginosa and S. aureus increased significantly. Conversely, level of reduced glutathione in (+)-catechin-treated E. coli, P. aeruginosa and S. aureus decreased significantly while glutathione disulfide increased significantly. Furthermore, malondialdehyde and fragmented DNA increased significantly following exposure to (+)-catechin. From the above findings, (+)-catechin enhanced the generation of reactive oxygen species (superoxide anion radical and hydroxyl radical) in E. coli, P. aeruginosa and S. aureus, possibly by autoxidation, Fenton chemistry and inhibiting electron transport chain resulting into lipid peroxidation and DNA fragmentation and consequentially bacterial cell death.

Standardized extract of Vitex doniana Sweet stalls protein oxidation, lipid peroxidation and DNA fragmention in acetaminophen-induced hepatotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Vitex doniana fruits are locally used in Nigeria as a remedy in the treatment of jaundice and liver related disease. The effect of methanolic extract of Vitex doniana fruits on acetaminophen induced protein oxidation, lipid peroxidation and DNA fragmentation was investigated in mice. MATERIALS AND METHODS: Antioxidant activity of the extract (0.2-1.0mg/mL) was investigated in vitro using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide ion, hydrogen peroxide, hydroxyl radical and ferric ion reducing system. Vitex doniana extract at 1.0mg/mL scavenged DPPH, superoxide ion, hydrogen peroxide, and hydroxyl radical by 86%, 78%, 80% and 72% respectively, it also reduced ferric ion significantly. Hepatoprotective effect of Vitex doniana fruits was monitored in acetaminophen-induced hepatotoxicity in mice. RESULTS: Acetaminophen-mediated alterations in serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin and total bilirubin levels in mice were significantly (P<0.05) attenuated by the extract. Similarly, acetaminophen-mediated decrease in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase was significantly (P<0.05) attenuated in the liver of mice. Increased levels of conjugated dienes, lipid hydroperoxides, malondialdehyde, protein carbonyl and fragmented DNA were significantly (P<0.05) lowered by methanolic extract of Vitex doniana fruits. CONCLUSIONS: Overall, the results of this study show that Vitex doniana fruits possess antioxidant properties and halted acetaminophen-mediated oxidative rout on cellular proteins, lipids and DNA, made possible by ß-sitosterol, platycodin D, apigenin, saikosaponin, chrysin and ellagitanin in the extract.

Whole Genome Amplification from Blood Spot Samples.

Whole genome amplification is an invaluable technique when working with DNA extracted from blood spots, as the DNA obtained from this source often is too limited for extensive genetic analysis. Two techniques that amplify the entire genome are common. Here, both are described with focus on the benefits and drawbacks of each system. However, in order to obtain the best possible WGA result the quality of input DNA extracted from the blood spot is essential, but also time consumption, flexibility in format and elution volume and price of the technology are factors influencing system choice. Here, three DNA extraction techniques are described and the above aspects are compared between the systems.

Cell-free DNA versus intact fetal cells for prenatal genetic diagnostics: what does the future hold?

Prenatal screening and diagnosis is currently focused on the development of a noninvasive prenatal diagnostic test capable of detecting abnormalities similar to those attainable with an invasive test. One contender is cell-free fetal DNA circulating in maternal plasma and the other is intact fetal cells either from the maternal blood or the cervix. Once adequate fetal DNA is available, laboratory analytic techniques, such as sequencing and microarray, can be applied allowing detection of most cytogenetic and Mendelian fetal disorders. The question is: how close are we to achieving this feat, and what does the future hold?

Hypoxia biomarkers, oxidative stress, and circulating microparticles in pediatric patients with thalassemia in Upper Egypt.

This study aimed to investigate the oxidative stress, hypoxia biomarkers, and circulating microparticles (MPs) in ß thalassemia major. The study included 56 children with thalassemia and 46 healthy controls. Hypoxia biomarkers, oxidative stress biomarkers, and total plasma fragmented DNA (fDNA) were detected by the standard methods. The MPs were assessed by flow cytometry. Hypoxia and oxidative stress biomarkers, fDNA, and MPs were higher and total antioxidant capacity (TAC) was lower in patients with thalassemia than the controls. In splenectomized patients and those who had complications, vascular endothelial growth factor (VEGF), malondialdehyde, fDNA, endothelial, platelet, and activated platelet MP levels were higher while, TAC was lower than the nonsplenectomized patients. In conclusion, the increased tissue hypoxia, oxidative stress in ß thalassemia, and its relationship with DNA damage and MPs release could explain many complications of thalassemia and may have therapeutic implications. The VEGF could serve as an important indicator for adequacy of blood transfusion in thalassemia.

Preparation of fragment libraries for next-generation sequencing on the applied biosystems SOLiD platform.

The primary purpose of this protocol is to prepare genomic DNA libraries that can then be analyzed by massively parallel next-generation sequencing on the Applied Biosystems SOLiD platform. This protocol can be adapted to next-generation sequencing workflows to ultimately generate up to 1 billion 50 bp sequence tags from the ends of each of the DNA molecules in the library in a single next-generation sequencing run.