Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae

With powerful genetics and a translucent cuticle, the Drosophila larva is an ideal model system for live imaging studies of neuronal cell biology and function. Here, we present an easy-to-use approach for high resolution live imaging in Drosophila using microfluidic chips. Two different designs allow for non-invasive and chemical-free immobilization of 3(rd) instar larvae over short (up to 1 hour) and long (up to 10 hours) time periods. We utilized these 'larva chips' to characterize several sub-cellular responses to axotomy which occur over a range of time scales in intact, unanaesthetized animals. These include waves of calcium which are induced within seconds of axotomy, and the intracellular transport of vesicles whose rate and flux within axons changes dramatically within 3 hours of axotomy. Axonal transport halts throughout the entire distal stump, but increases in the proximal stump. These responses precede the degeneration of the distal stump and regenerative sprouting of the proximal stump, which is initiated after a 7 hour period of dormancy and is associated with a dramatic increase in F-actin dynamics. In addition to allowing for the study of axonal regeneration in vivo, the larva chips can be utilized for a wide variety of in vivo imaging applications in Drosophila.     

Glutathione S-transferase M1, T1 and P1 polymorphisms and bladder cancer risk in Egyptians

Previous studies suggest that bladder cancer risk may vary with GST genotype but these results are inconsistent. The aim of this study was to explore whether GSTM1, GSTT1 and GSTP polymorphisms were associated with increased bladder cancer risk in an Egyptian population. GSTM1, GSTT1 and GSTP1 genotype frequencies were determined in bladder cancer cases (n=72) and healthy controls with no history of malignancies (n=82) using PCR-based techniques. The GSTT1*2 genotype was particularly associated with increased risk (OR 2.71, 95%CI 1.27-5.73) and the GSTM1*2 genotype to a lesser extent (OR 1.63, 95%CI 0.85-3.10). 18.1% of cases but only 7.3% of controls were GSTP1*B*B homozygotes (OR 2.38, 95%CI 0.83-6.87). The presence of two or more a priori at-risk genotypes was associated with increased bladder cancer risk (OR 2.42; 95%CI 1.47-3.97). These results suggest that polymorphisms in the GST genes are associated with increased risk of bladder cancer among Egyptians.     

Study by transmission and scanning electron microscopy of the morphogenesis of three types of lingual papillae in the albino rat (Rattus rattus)

El‐Bakry, A.M. 2010. Study by transmission and scanning electron microscopy of the morphogenesis of three types of lingual papillae in the albino rat (Rattus rattus).—Acta Zoologica (Stockholm) 91 : 267–278 Tongues were removed from albino rat foetus on days 12 (E12) and 16 (E16) of gestation and from newborns (P0) and from juvenile rats on days 7 (P7), 14 (P14) and 21 (P21) postnatally for investigation by light, scanning, and transmission electron microscopy. Significant changes appeared during the morphogenesis of the papillae. At E12, two rows of rudiments of fungiform papillae were extended bilaterally on the anterior half of the tongue. At E16, the rudiments of fungiform papillae were regularly arranged in a lattice‐like pattern. A rudiment of circumvallate papillae could be recognized. No rudiment of filiform papillae was visible. No evidence of keratinization was recognizable. At P0, rudiments of filiform papillae were visible but had a more rounded appearance, with keratinization. The fungiform and circumvallate papillae were large and their outlines were somewhat irregular as that found in the adult rat. At P7, the filiform papillae were large and slender. The fungiform papillae became large and the shape of circumvallate papillae was almost similar to that observed in the adult. At P14 and P21, the shape and structure of the three types of papillae were irregular as those found in the adult. In conclusion, the rudiments of the fungiform and circumvallate papillae were visible earlier than those of the filiform papillae. The morphogenesis of filiform papillae advanced in a parallel manner with the keratinization of the lingual epithelium, in the period from just before birth to a few weeks after birth.     

HIV treatment models with time delay

The present paper shows possible effects of antiretroviral treatment on the dynamics of the spread of the disease of human immunodeficiency virus infection in a population of varying size. By introducing time delays, we model the latency period and the delayed onset of positive treatment effects in the patients. The Hopf bifurcation and stability behaviour of the delay differential-equation model are analysed and simulations for different scenarios depending on the size of the treatment-induced delay are presented, and the results are discussed in detail.     

Multiplex Pyrosequencing

We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.     

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

Epilepsy seizure detection using eigen-system spectral estimation and Multiple Layer Perceptron neural network

In this paper, a new approach based on eigen-systems pseudo-spectral estimation methods, namely Eigenvector (EV) and MUSIC, and Multiple Layer Perceptron (MLP) neural network is introduced. In this approach, the calculated EEG (electroencephalogram) spectrum is divided into smaller frequency sub-bands. Then, a set of features, {maximum, entropy, average, standard deviation, mobility}, are extracted from these sub-bands. Next, incorporating a set of the EEG time domain features {standard deviation, complexity measure} with the spectral feature set, a feature vector is formed. The feature vector is then fetched into a MLP neural network to classify the signal into the following three states: normal (healthy), epileptic patient signal in a seizure-free interval (inter-ictal), and epileptic patient signal in a full seizure interval (ictal). The experimental results show that the classification of the EEG signals maybe achieved with approximately 97.5% accuracy and the variance of 0.095% using an available public EEG signals database. The results are among the best reported methods for classifying the three states aforementioned. This is a high speed with high accuracy as well as low misclassifying rate method so it can make the practical and real-time detection of this chronic disease feasible.     

Patterns and Determinants of Halophilic Archaea (Class Halobacteria) Diversity in Tunisian Endorheic Salt Lakes and Sebkhet Systems

We examined the diversity and community structure of members of the halophilic Archaea (class Halobacteria) in samples from central and southern Tunisian endorheic salt lakes and sebkhet (also known as sebkha) systems using targeted 16S rRNA gene diversity survey and quantitative PCR (qPCR) approaches. Twenty-three different samples from four distinct locations exhibiting a wide range of salinities (2% to 37%) and physical characteristics (water, salt crust, sediment, and biofilm) were examined. A total of 4,759 operational taxonomic units at the 0.03 (species-level) cutoff (OTU_0.03s) belonging to 45 currently recognized genera were identified, with 8 to 43 genera (average, 30) identified per sample. In spite of the large number of genera detected per sample, only a limited number (i.e., 2 to 16) usually constituted the majority (≥80%) of encountered sequences. Halobacteria diversity showed a strong negative correlation to salinity (Pearson correlation coefficient = −0.92), and community structure analysis identified salinity, rather than the location or physical characteristics of the sample, as the most important factor shaping the Halobacteria community structure. The relative abundance of genera capable of biosynthesis of the compatible solute(s) trehalose or 2-sulfotrehalose decreased with increasing salinities (Pearson correlation coefficient = −0.80). Indeed, qPCR analysis demonstrated that the Halobacteria otsB (trehalose-6-phosphatase)/16S rRNA gene ratio decreases with increasing salinities (Pearson correlation coefficient = −0.87). The results highlight patterns and determinants of Halobacteria diversity at a previously unexplored ecosystem and indicate that genera lacking trehalose biosynthetic capabilities are more adapted to growth in and colonization of hypersaline (>25% salt) ecosystems than trehalose producers.