Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects

BACKGROUND AND OBJECTIVE:

Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario.

MATERIALS AND METHODS:

A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup.

RESULTS:

Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals.

CONCLUSION:

Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India.     

Physiological and biochemical consequences of host-plasmid interaction--a case study with Corynebacterium renale, a multiple cryptic plasmid containing strain

Corynebacterium renale harbors four small cryptic plasmids, pCR1, pCR2, pCR3 and pCR4, and can be a good system for understanding host-plasmid interactions. In the present study, effect of plasmid loss and their subsequent introduction on various properties of the host was evaluated. Loss of plasmids caused a reduction in bacterial size and also slowed down their growth rate, μ, and respiratory rate, r. Both μ and r values were partially recovered in C. renale R, obtained by retransformation of the cured strain with all the four cryptic plasmids. Further delineation revealed that a 3153bp plasmid pCR2 alone is sufficient for the observed increase in μ in C. renale R. The advantages conferred by the remaining three plasmids may be are two subtle to be seen under laboratory conditions. Overall, the observations point to the gross metabolic crisis in the host partly as a result of loss of plasmids. Based on the findings, a mutualistic relationship between the host and the plasmids resulting from their coevolution is proposed.     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Hypo-osmotic swelling modifies glutamate-glutamine cycle in the cerebral cortex and in astrocyte cultures

In our previous work, we found that perfusion of the rat cerebral cortex with hypo-osmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R. E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels, whereas mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypo-osmotic medium to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. As the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypo-osmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[(3) H]glutamine by ～twofold and produced no changes in L-[(3) H]glutamine uptake. In comparison, hypo-osmotic medium stimulated the release of glutamate (traced with D-[(3) H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypo-osmotic medium caused a 20% inhibition of astrocytic conversion of L-[(3) H]glutamate into L-[(3) H]glutamine by glutamine synthetase. Using an HPLC assay, we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (i) inhibits astrocytic glutamine synthetase activity, and (ii) reduces substrate availability for this enzyme because of the activation of volume-regulated anion channels. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia.     

Evaluation of Solanum xanthocarpum extract as a synergist for cypermethrin against larvae of the filarial vector Culex quinquefasciatus (Say)

Cypermethrin and crude extracts of Solanum xanthocarpum were both observed for their larvicidal activity against Culex quinquefasciatus. Petroleum ether extract with lethal concentration (LC)₅₀ and LC₉₀ of 41.28 and 111.16 p.p.m. after 24 h and LC₅₀ 38.48 and LC₉₀ 80.83 p.p.m. after 48 h, respectively, was found to be the most effective, followed by carbon tetrachloride and methanol extracts. LC₅₀ and LC₉₀ for cypermethrin were 0.0027 and 0.0097 p.p.m. after 24 h and 0.0013 and 0.0092 p.p.m. after 48 h of exposure, respectively. Combined formulations were evaluated for synergistic activity and a 1:1 ratio of cypermethrin and petroleum ether extract was observed to be more effective than 1:2 and 1:4 ratios. Combinations of S. xanthocarpum extracts and cypermethrin demonstrated higher larvicidal activity, indicating synergistic activity. These results demonstrate the need for further studies on the effectiveness and toxicity to humans and animals, particularly aquatic forms.