A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR.

OBJECTIVES: To study the ability of a stabilizing reagent to prevent cellular DNA contamination of cell-free DNA (cfDNA) in plasma during whole blood sample storage and shipping. DESIGN AND METHODS: Samples were drawn from healthy donors into K3EDTA and Cell-Free DNA BCTs (BCT) and stored at room temperature (RT). Aliquots were removed at specified time points and cfDNA was purified from the plasma. A Droplet Digital PCR (ddPCR) assay that amplifies a short ß-actin gene fragment (136 bp) was used to measure the total plasma cfDNA (pDNA) concentration while a longer ß-actin fragment (420 bp) was used to quantify genomic DNA (gDNA). In a follow-up experiment, blood samples drawn into the same types of tubes were shipped round trip by overnight air before cfDNA was isolated and analyzed. RESULTS: Blood stored in K3EDTA tubes at RT showed increases in pDNA and gDNA concentrations over time. However, both pDNA and gDNA levels remained stable in BCT for at least seven days. On day 14, there was a 4.5-fold increase in pDNA in BCT as compared to >200-fold increase in K3EDTA tubes. Likewise, gDNA increased <2-fold on day 14 in BCT as opposed to a 456-fold increase in K3EDTA tubes. Similar results were observed after samples were shipped. CONCLUSIONS: Cell-Free DNA BCTs prevent gDNA contamination that may occur due to nucleated cell disruption during sample storage and shipping. This novel blood collection tube provides a method for obtaining stable cfDNA samples for rare target detection and accurate analysis while mitigating the threat of gDNA contamination.

A new methodology to preserve the original proportion and integrity of cell-free fetal DNA in maternal plasma during sample processing and storage.

OBJECTIVE: To develop a standardized blood collection device that preserves fetal cell-free DNA and minimizes the cell-free DNA background in maternal plasma. METHODS: Blood samples were drawn from healthy pregnant donors into K(3)EDTA (BD vacutainer) and Cell-free DNA BCT tubes (Streck, Inc.) and kept at ambient temperature. Plasma was separated by centrifugation and cell-free DNA was extracted. Cell-free DNA from plasma was quantified by quantitative real-time polymerase chain reaction. RESULTS: Blood drawn into Cell-free DNA BCT tubes showed no change in the original proportion of fetal cell-free DNA during a 14-day storage period at ambient temperature. Conversely, maternal blood drawn into K(3)EDTA tubes showed a steady reduction in the original proportion of fetal cell-free DNA over the same time period. Using maternal plasma stored in Cell-free DNA BCT tubes for 14 days, fetal cell-free DNA was amplified 80-fold using whole genome amplification (WGA). CONCLUSION: Using Streck's Cell-free DNA BCT tubes, it is possible to preserve the original proportion of fetal cell-free DNA for extended times as well as minimize the post-sampling maternal cell-free DNA background. Preserved in this way, fetal cell-free DNA can be amplified by WGA technology to be used in prenatal diagnostic tests.

Doença do nível adjacente após artrodese da coluna lombar: revisão da literatura / Adjacent segment disease after lumbar spine fusion: literature review

A degeneração do nível adjacente acima ou abaixo da fusão na coluna lombar é definida como doença do nível adjacente. Atualmente é considerada como uma complicação tardia e freqüente. Do ponto de vista radiográfico apresenta uma incidência de 5,2 a 100%. No entanto a incidência de pacientes sintomáticos é menor,variando de 5,2 a 18,5%. A etiologia é incerta. Provavelmente a origem está no aumento da pressão intradiscal,no aumento da carga facetária e no aumento da mobilidade. Os potenciais fatores de risco incluem: instrumentação, tamanho de fusão, mau alinhamento sagital, lesão facetária, idade e degeneração pré-existente. Otratamento pode ser clínico ou cirúrgico. O tratamento cirúrgico consiste na descompressão dos elementos neurais e extensão da fusão. Os resultados após a nova cirurgia são modestos.

Psychosis and substance abuse: cause, effect or coincidence?

An association between substance abuse and major psychiatric illness is increasingly well recognised, but most studies have been conducted in the USA and have focussed upon patients with schizophrenia rather than other disorders. We conducted a survey of 38 consecutively admitted patients with DSM-III-R functional psychoses. A semi-structured substance abuse interview was administered and a urine specimen for drug metabolite screening requested. The prevalence of cigarette smoking (63%) and current illicit drug use (26%) were higher than general population norms. The 16 subjects with schizophrenia and related disorders were more likely to smoke cigarettes than the 22 patients with an affective disorder (p = 0.008, odds ratio 8.4, 95% Cl 1.3-69.6), and showed tendencies to more illicit drug and alcohol consumption. Illicit drug users were more likely to have a forensic history and less likely to have entered further education. Substance abuse is common among patients with psychoses, particularly in those with schizophrenia and related disorders. All psychotic patients should have a detailed drug history taken, and therapeutic attempts made to reduce consumption.

Possible differences in the regenerative roles played by thioltransferase and thioredoxin for oxidatively damaged proteins.

A possible involvement of thioltransferase (also known as glutaredoxin) in the regenerative reaction of proteins inactivated by oxidative stress were examined in vitro using the enzyme purified from bovine liver. Thioltransferase at physiological concentrations, together with glutathione, glutathione reductase and NADPH, regenerated the oxidatively damaged proteins with a comparable potency to that of thioredoxin. Experiments performed with protein substrates with their critical cysteine residues oxidized differently, that is, phosphofruktokinase and glyceraldehyde 3-phosphate dehydrogenase with mixed disulfide bonds and glyceraldehyde 3-phosphate dehydrogenase with sulfenyl or sulfinyl groups, indicated that thioltransferase regenerated the proteins inactivated by mixed disulfide formation more efficiently than thioredoxin, whereas thioredoxin preferentially regenerated the proteins inactivated by monothiol oxidation to sulfenic or sulfinic acid. These findings suggested that thioltransferase exerted regenerative effects on oxidatively damaged proteins like its cognate protein, thioredoxin, but with different substrate specificity, and their relative contribution to the regeneration reaction is dependent on the form of the oxidized thiols of the damaged proteins.

Cloning and sequencing of the cDNA encoding human glutaredoxin.

Glutaredoxin (thioltransferase) is a small, heat-stable protein, which is involved in thiol/disulfide exchange reactions. We have isolated a cDNA that encodes glutaredoxin from a human brain cDNA library. The encoded protein contains 106 amino acids with a calculated molecular mass of 11.76 kDa and an isoelectric point of 8.09. The amino acid sequence deduced from the cDNA is more than 80% identical to those of other mammalian glutaredoxins.

Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2-14 microM) of reduced thioredoxin, with 0.125 microM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37 degrees C. Although the treatment of endothelial cells with 0.2-12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 degrees C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.

Effect of Artocarpus heterophyllus and Asteracanthus longifolia on glucose tolerance in normal human subjects and in maturity-onset diabetic patients.

Investigations were carried out to evaluate the effects of hot-water extracts of Artocarpus heterophyllus leaves and Asteracanthus longifolia whole plant material on the glucose tolerance of normal human subjects and maturity-onset diabetic patients. The extracts of both Artocarpus heterophyllus and Asteracanthus longifolia significantly improved glucose tolerance in the normal subjects and the diabetic patients when investigated at oral doses equivalent to 20 g/kg of starting material.

Hypoglycaemic activity of some medicinal plants in Sri-Lanka.

1. Investigations were carried out to determine whether aqueous extracts of Osbeckia octandra, Artocarpus heterophyllus and Bambusa vulgaris truly possess oral hypoglycaemic activity. 2. All three plant extracts significantly lowered the fasting blood glucose level and markedly improved glucose tolerance in Sprague-Dawley rats. 3. A maximum hypoglycaemic activity was observed at +3 hr with O. octandra and B. vulgaris; with A. heterophyllus a maximum effect was not observed even at +5 hr. 4. The hypoglycaemic activity of O. octandra was comparable with that of tolbutamide while that of A. heterophyllus or B. vulgaris was better than that of tolbutamide. 5. The magnitude of the hypoglycaemic effects varied with the dosage used and the time of storage (except with A. heterophyllus, whose activity did not change with storage even up to 3 days).

A preliminary investigation of the possible hypoglycaemic activity of Asteracanthus longifolia.

Investigations were carried out to confirm or otherwise disprove the view held by many Ayurvedic and other traditional medical practitioners in Sri Lanka, that Asteracanthus longifolia possesses hypoglycaemic properties. The effects of an aqueous extract of the whole plant on fasting blood glucose level and glucose tolerance were investigated using Sprague-Dawley rats. The results indicate that aqueous extracts of A. longifolia can significantly lower the fasting blood glucose level and markedly improve the glucose tolerance of the rats. The hypoglycaemic effect produced by a therapeutic dose (equivalent to 5 g/kg of starting material) was comparable to that produced by a therapeutic dose (15 mg/kg of tolbutamide. The magnitude of the hypoglycaemic effect was found to vary with the dosage administered and the storage time of the prepared extract.