Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Variants of ST8SIA1 are associated with risk of developing multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system of unknown etiology with both genetic and environmental factors playing a role in susceptibility. To date, the HLA DR15/DQ6 haplotype within the major histocompatibility complex on chromosome 6p, is the strongest genetic risk factor associated with MS susceptibility. Additional alleles of IL7 and IL2 have been identified as risk factors for MS with small effect. Here we present two independent studies supporting an allelic association of MS with polymorphisms in the ST8SIA1 gene, located on chromosome 12p12 and encoding ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. The initial association was made in a single three-generation family where a single-nucleotide polymorphism (SNP) rs4762896, was segregating together with HLA DR15/DQ6 in MS patients. A study of 274 family trios (affected child and both unaffected parents) from Australia validated the association of ST8SIA1 in individuals with MS, showing transmission disequilibrium of the paternal alleles for three additional SNPs, namely rs704219, rs2041906, and rs1558793, with p = 0.001, p = 0.01 and p = 0.01 respectively. These findings implicate ST8SIA1 as a possible novel susceptibility gene for MS.     

Alternative splicing and protein function

Background  

Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.     

Potential role of viruses in white plague coral disease

White plague (WP)-like diseases of tropical corals are implicated in reef decline worldwide, although their etiological cause is generally unknown. Studies thus far have focused on bacterial or eukaryotic pathogens as the source of these diseases; no studies have examined the role of viruses. Using a combination of transmission electron microscopy (TEM) and 454 pyrosequencing, we compared 24 viral metagenomes generated from Montastraea annularis corals showing signs of WP-like disease and/or bleaching, control conspecific corals, and adjacent seawater. TEM was used for visual inspection of diseased coral tissue. No bacteria were visually identified within diseased coral tissues, but viral particles and sequence similarities to eukaryotic circular Rep-encoding single-stranded DNA viruses and their associated satellites (SCSDVs) were abundant in WP diseased tissues. In contrast, sequence similarities to SCSDVs were not found in any healthy coral tissues, suggesting SCSDVs might have a role in WP disease. Furthermore, Herpesviridae gene signatures dominated healthy tissues, corroborating reports that herpes-like viruses infect all corals. Nucleocytoplasmic large DNA virus (NCLDV) sequences, similar to those recently identified in cultures of Symbiodinium (the algal symbionts of corals), were most common in bleached corals. This finding further implicates that these NCLDV viruses may have a role in bleaching, as suggested in previous studies. This study determined that a specific group of viruses is associated with diseased Caribbean corals and highlights the potential for viral disease in regional coral reef decline.     

Comparison of multimarker logistic regression models, with application to a genomewide scan of schizophrenia

Background

Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue.

Results

Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%).

Conclusions

Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.     

Augmentation of partially regenerated nerves by end-to-side side-to-side grafting neurotization: experience based on eight late obstetric brachial plexus cases

Objective

The effect of end-to-side neurotization of partially regenerated recipient nerves on improving motor power in late obstetric brachial plexus lesions, so-called nerve augmentation, was investigated.

Methods

Eight cases aged 3 – 7 years were operated upon and followed up for 4 years (C5,6 rupture C7,8T1 avulsion: 5; C5,6,7,8 rupture T1 avulsion:1; C5,6,8T1 rupture C7 avulsion:1; C5,6,7 ruptureC8 T1 compression: one 3 year presentation after former neurotization at 3 months). Grade 1–3 muscles were neurotized. Grade0 muscles were neurotized, if the electromyogram showed scattered motor unit action potentials on voluntary contraction without interference pattern. Donor nerves included: the phrenic, accessory, descending and ascending loops of the ansa cervicalis, 3^rd and 4^th intercostals and contralateral C7.

Results

Superior proximal to distal regeneration was observed firstly. Differential regeneration of muscles supplied by the same nerve was observed secondly (superior supraspinatus to infraspinatus regeneration). Differential regeneration of antagonistic muscles was observed thirdly (superior biceps to triceps and pronator teres to supinator recovery). Differential regeneration of fibres within the same muscle was observed fourthly (superior anterior and middle to posterior deltoid regeneration). Differential regeneration of muscles having different preoperative motor powers was noted fifthly; improvement to Grade 3 or more occurred more in Grade2 than in Grade0 or Grade1 muscles. Improvements of cocontractions and of shoulder, forearm and wrist deformities were noted sixthly. The shoulder, elbow and hand scores improved in 4 cases.

Limitations

The sample size is small. Controls are necessary to rule out any natural improvement of the lesion. There is intra- and interobserver variability in testing muscle power and cocontractions.

Conclusion

Nerve augmentation improves cocontractions and muscle power in the biceps, pectoral muscles, supraspinatus, anterior and lateral deltoids, triceps and in Grade2 or more forearm muscles. As it is less expected to improve infraspinatus power, it should be associated with a humeral derotation osteotomy and tendon transfer. Function to non improving Grade 0 or 1 forearm muscles should be restored by muscle transplantation.

Level of evidence

Level IV, prospective case series.     

Ratio-dependent parasitism with Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) on egg rafts of Nezara viridula (Linnaeus) (Hemiptera: Pentatomidae): effect of experimental variables and compatibility of 'ratio' and 'Holling' models

Abstract Egg rafts of Nezara viridula were exposed to the parasitoid wasp Trissolcus basalis in experimental arenas to establish the relationship of the rates of attack and parasitism to various combinations of arena size, parasitoid density, host density and parasitoid-to-host ratio. Arena sizes were varied in the ratio 1:9:63, with the largest having a search area of 1.44 m². Parasitoid and host densities were varied over a 27-fold range. The parasitoid-to-host ratios used were 1:1, 3:1 and 6:1. Finding time was related to a constant factor (flight propensity), rather than to the difficulty of finding (density of hosts). Initial attack rates were therefore related only to parasitoid numbers (or density), even at the lower densities and ratios. Parasitism rates (a function of attack rate per host) were thus also strongly related to parasitoid to host ratio, regardless of densities used and arena sizes. Even reducing host density, while keeping time and parasitoid density constant, increased the parasitism rate. A ratio model for parasitism rate was therefore compatible with the data but the more explicit Holling 'disc' equation was also compatible because handling time was sufficiently large to make it sensitive to the ratio of parasitoids to hosts for the densities used. We conclude that the two models would predict different results if the density of host egg rafts was in a range below one per square metre.     

ER Stress Induced by Tunicamycin Triggers α-Synuclein Oligomerization,Dopaminergic Neurons Death and Locomotor Impairment: a New Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons of the substantia nigra pars compacta (SNpc), leading to the major clinical abnormalities that characterize this disease. Although PD’s etiology is unknown, α-synuclein aggregation plays a pivotal role in PD pathogenesis, which could be associated to some pathological processes such as oxidative stress, endoplasmic reticulum (ER) stress, impaired protein degradation, and mitochondrial dysfunction. Increasing experimental evidence indicates that ER stress is involved in PD, however most of the described results employed cultured cell lines and genetically modified animal models. In this study, we developed a new ER stress rat model employing the well-known ER stressor tunicamycin (Tm). To evaluate if ER stress was able to induce PD features, we performed an intranigral injection of Tm (0.1 μg/cerebral hemisphere) and animals (male Wistar rats) were analyzed 7 days post injection. The classical 6-OHDA neurotoxin model (1 μg/cerebral hemisphere) was used as an established positive control for PD. We show that Tm injection induced locomotor impairment, dopaminergic neurons death, and activation of astroglia. In addition, we observed an extensive α-synuclein oligomerization in SNpc of Tm-injected animals when compared with DMSO-injected controls. Finally, both Tm and 6-OHDA treated animals presented increased levels of ER stress markers. Taken together, these findings show for the first time that the ER stressor Tm recapitulates some of the phenotypic characteristics observed in rodent models of PD, reinforcing the concept that ER stress could be an important contributor to the pathophysiology of PD. Therefore, we propose the intranigral Tm injection as a new ER stress-based model for the study of PD in vivo.