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.     

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.     

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.     

The neuronal adaptor protein X11alpha reduces Abeta levels in the brains of Alzheimer's APPswe Tg2576 transgenic mice

Increased production and deposition of the 40-42-amino acid beta-amyloid peptide (Abeta) is believed to be central to the pathogenesis of Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP), but the mechanisms that regulate APP processing to produce Abeta are not fully understood. X11alpha (also known as munc-18-interacting protein-1 (Mint1)) is a neuronal adaptor protein that binds APP and modulates APP processing in transfected non-neuronal cells. To investigate the in vivo effect of X11alpha on Abeta production in the brain, we created transgenic mice that overexpress X11alpha and crossed these with transgenics harboring a familial Alzheimer's disease mutant APP that produces increased levels of Abeta (APPswe Tg2576 mice). Analyses of Abeta levels in the offspring generated from two separate X11alpha founder mice revealed a significant, approximate 20% decrease in Abeta(1-40) in double transgenic mice expressing APPswe/X11alpha compared with APPswe mice. At a key time point in Abeta plaque deposition (8 months old), the number of Abeta plaques was also deceased in APPswe/X11alpha mice. Thus, we report here the first demonstration that X11alpha inhibits Abeta production and deposition in vivo in the brain.     

Capture and commercialization of blue land crabs ("guaiamum") Cardisoma guanhumi (Lattreille, 1825) along the coast of Bahia State, Brazil: an ethnoecological approach

Background

Blue Land Crab (Cardisoma guanhumi) is one of the most important crustacean species captured and commercialized in Brazil. Although this species is not considered to be threatened with extinction, populations of C. guanhumi are known to be rapidly diminishing due to heavy harvesting pressures and degradation of their natural habitats, highlighting the necessity of developing and implanting management and protection strategies for their populations. There have been no ethnozoological publications that have focused specifically on C. guanhumi, in spite of importance of this type of information for developing efficient management plans of resource utilization. So, the present work describes the ethnoecological aspects of the capture and commercialization of C. guanhumi by a fishing community in northeastern Brazil.

Methods

Field work was carried out in the municipality of Mucuri, Bahia in Brazil, between the months of January and March/2011 through the use of open semi-structured interviews with all of the crustacean harvesters in city who acknowledged their work in capturing this species, totaling 12 interviewees. The informants were identified through the use of the "snowball" sampling technique. In addition to the interviews themselves, the "guided tour" technique and direct observations was employed.

Results

According all the interviewees, the C. guanhumi is popularly called "guaiamum" and is collected in "apicum" zones. They recognize sexual dimorphism in the species based on three morphological characteristics and the harvesters also pointed two stages in the reproductive cycle during the year and another phase mentioned by the interviewees was ecdysis. All of the interviewed affirmed that the size and the quantities C. guanhumi stocks in Mucuri have been diminishing. All of the interviewees agreed that the species and other mangrove resources constituted their principal source of income. The harvesters dedicated three to five days a week to collect Blue Land Crabs and the principal technique utilized for capturing is a trap called a "ratoeira" (rat-trap).

Conclusions

The results of the present work demonstrated that the community retains a vast and important volume of knowledge about C. guanhumi that could subsidize both scientific studies and the elaboration of viable management and conservation strategies for this species.     

Antenatal breast expression in women with diabetes: outcomes from a retrospective cohort study

Background

Women with diabetes are sometimes advised to express breast milk antenatally to prepare for breastfeeding and to store colostrum for infant feeding in preventing or treating hypoglycaemia after the birth. The acceptability, risks and benefits of this practice have not been evaluated. This was aimed to investigate the pattern of antenatal breast expression uptake and its relationship with birth outcomes in women with diabetes.

Methods

This was part of a two year retrospective cohort study of pregnant women with diabetes (type 1, 2 and gestational diabetes) who gave birth during 2001–2003 in Derby Hospitals NHS Foundation Trust (n = 94). The information on the practice of antenatal breastfeeding expression and birth outcomes was collected via self-administered questionnaires and by examining maternity records.

Results

Thirty-seven percent of women (35/94) recalled that they were advised to express antenatally and 17% did (16/94). The mean gestational age at birth for women who hand-expressed was lower than that for those who did not (mean difference (MD) (95% confidence intervals (CI)): -1.2 (?2.4 to 0.04), p = 0.06). A higher proportion of babies from the antenatal expression group were admitted to special care baby units (SCBU) (MD (95% CI): 21% (?3.9 to 46.3).

Conclusions

Less than half the women who stated that they were advised to express, did so. There seems to be an indication that antenatal breast milk expression and lower gestational age at birth are associated. The trend of a higher rate of SCBU admission for babies from the breast milk expression group compared to those who did not express antenatally is of concern. An appropriately-powered randomised controlled trial is needed to determine the safety of this practice and its acceptability to women and health professionals before it can be recommended for implementation in practice.