The effect of sample handling on cross sectional HIV incidence testing results

Objective(s)

To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays.

Methods

Serum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4°C, 25°C and 37°C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay).

Results

Compared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4°C or 25°C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37°C prior to testing.

Conclusions

Samples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4°C or 25°C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays.     

Regulation of glutathione S-transferases of Zea mays in plants and cell cultures

An antiserum to glutathione S-transferase (EC 2.5.1.18) from maize (Zea mays L.) responsible for herbicide detoxification has been raised in rabbit. The antiserum was specific to the M_r 26000 subunit of the enzyme from maize seedlings and suspension-cultured cells, and recognized the isoenzymes active toward both atrazine and metolachlor. When plants were treated for 24 h with the herbicide antidote N,N-diallyl-2-2-dich-loroacetamide (DDCA), enzyme activities toward metolachlor were doubled in the roots and this was associated with a 70% increase in immunodetectable protein. Translation of polysomal RNA in vitro showed that the increase in the transferase in root tissue was brought about by a ninefold increase in mRNA activity encoding the enzyme. Treatment of suspension-cultured cells with cinnamic acid, metolachlor and DDCA raised enzyme activities but did not increase synthesis of glutathione S-transferase. In cultured maize cells, enzyme synthesis was maximal in mid-logarithmic phase, coinciding with the highest levels of enzyme activity. When callus cultures were established from the shoots of a maize line known to conjugate chloro-s-triazines, enzyme activity towards atrazine was lost during primary dedifferentiation. However, levels of total immunodetectable enzyme and activity toward metolachlor were increased in cultured cells compared with the parent shoot tissue.     

Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.     

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.     

Identification of Genes Transcriptionally Responsive to the Loss of MLL Fusions in MLL-Rearranged Acute Lymphoblastic Leukemia

Introduction

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions.

Methods

For this, we performed gene expression profiling after siRNA-mediated repression of MLL-AF4, MLL-ENL, and AF4-MLL in MLL-rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples.

Results

Genes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were affected in response to the repression of AF4-MLL showed significant enrichment in gene expression profiles associated with AF4-MLL expressing t(4;11)+ infant ALL patient samples.

Conclusion

We conclude that the here identified genes readily responsive to the loss of MLL fusion expression potentially represent attractive therapeutic targets and may provide additional insights in MLL-rearranged acute leukemias.     

Phenotypic plasticity in chemical defence of butterflies allows usage of diverse host plants

Host plant specialization is a major force driving ecological niche partitioning and diversification in insect herbivores. The cyanogenic defences of Passiflora plants keep most herbivores at bay, but not the larvae of Heliconius butterflies, which can both sequester and biosynthesize cyanogenic compounds. Here, we demonstrate that both Heliconius cydno chioneus and H. melpomene rosina have remarkable plasticity in their chemical defences. When feeding on Passiflora species with cyanogenic compounds that they can readily sequester, both species downregulate the biosynthesis of these compounds. By contrast, when fed on Passiflora plants that do not contain cyanogenic glucosides that can be sequestered, both species increase biosynthesis. This biochemical plasticity comes at a fitness cost for the more specialist H. m. rosina, as adult size and weight for this species negatively correlate with biosynthesis levels, but not for the more generalist H. c. chioneus. By contrast, H. m rosina has increased performance when sequestration is possible on its specialized host plant. In summary, phenotypic plasticity in biochemical responses to different host plants offers these butterflies the ability to widen their range of potential hosts within the Passiflora genus, while maintaining their chemical defences.     

Variants of the <Emphasis Type="Italic">EAAT2</Emphasis> Glutamate Transporter Gene Promoter Are Associated with Cerebral Palsy in Preterm Infants

Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200C>A and g.-181A>C) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns’ dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A alleles at both loci appeared to be detrimental and consequently, the risk of developing cerebral palsy increased four- and sixfold for each additional detrimental allele at -200 and -181 bp, respectively. The two SNPs altered the regulation of the EAAT2 promoter activity and glutamate homeostasis. This study highlights the significance of glutamate in the pathogenesis of preterm brain injury and subsequent development of cerebral palsy and neurodevelopmental disabilities. Furthermore, the described EAAT2 SNPs may be an early biomarker of vulnerability to neurodisability and may aid the development of targeted treatment strategies.