bluuebunny/crossref_metadata_2025 huggingface dataset:
Independent inactivation of arginine decarboxylase genes by nonsense and missense mutations led to pseudogene formation in Chlamydia trachomatisserovar L2 and D strains
Authors: Giles Teresa N , Fisher Derek J , Graham David E
Published: July 2009

Background

Chlamydia have reduced genomes that reflect their obligately parasitic lifestyle. Despite their different tissue tropisms, chlamydial strains share a large number of common genes and have few recognized pseudogenes, indicating genomic stability. All of theChlamydiaceaehave homologs of theaaxABCgene cluster that encodes a functional arginine:agmatine exchange system inChlamydia(Chlamydophila)pneumoniae. However,Chlamydia trachomatisserovar L2 strains have a nonsense mutation in theiraaxBgenes, andC. trachomatisserovar A and B strains have frameshift mutations in theiraaxChomologs, suggesting that relaxed selection may have enabled the evolution ofaaxpseudogenes. Biochemical experiments were performed to determine whether theaaxABCgenes fromC. trachomatisstrains were transcribed, and mutagenesis was used to identify nucleotide substitutions that prevent protein maturation and activity. Molecular evolution techniques were applied to determine the relaxation of selection and the scope ofaaxgene inactivation in theChlamydiales.

Results

TheaaxABCgenes were co-transcribed inC. trachomatisL2/434, during the mid-late stage of cellular infection. However, a stop codon in theaaxBgene from this strain prevented the heterologous production of an active pyruvoyl-dependent arginine decarboxylase. Replacing that ochre codon with its ancestral tryptophan codon rescued the activity of this self-cleaving enzyme. TheaaxBgene fromC. trachomatisD/UW-3 was heterologously expressed as a proenzyme that failed to cleave and form the catalytic pyruvoyl cofactor. This inactive protein could be rescued by replacing the arginine-115 codon with an ancestral glycine codon. TheaaxCgene from the D/UW-3 strain encoded an active arginine:agmatine antiporter protein, while the L2/434 homolog was unexpectedly inactive. Yet the frequencies of nonsynonymous versus synonymous nucleotide substitutions show no signs of relaxed selection, consistent with the recent inactivation of these genes.

Conclusion

The ancestor of theChlamydiaceaehad a functional arginine:agmatine exchange system that is decaying through independent, parallel processes in theC. trachomatislineage. Differences in arginine metabolism amongChlamydiaceaespecies may be partly associated with their tissue tropism, possibly due to the protection conferred by a functional arginine-agmatine exchange system against host nitric oxide production and innate immunity. The independent loss of AaxB activity in all sequencedC. trachomatisstrains indicates continual gene inactivation and illustrates the difficulty of recognizing recent bacterial pseudogenes from sequence comparison, transcriptional profiling or the analysis of nucleotide substitution rates.
Current genetic differentiation of Coffea canephoraPierre ex A. Froehn in the Guineo-Congolian African zone: cumulative impact of ancient climatic changes and recent human activities
Authors: Gomez Céline , Dussert Stéphane , Hamon Perla , Hamon Serge , de Kochko Alexandre , Poncet Valérie
Published: July 2009

Background

Among Coffea species, C. canephora has the widest natural distribution area in tropical African forests. It represents a good model for analyzing the geographical distribution of diversity in relation to locations proposed as part of the "refuge theory". In this study, we used both microsatellite (simple sequence repeat, SSR) and restriction fragment length polymorphism (RFLP) markers to investigate the genetic variation pattern of C. canephora in the Guineo-Congolean distribution zone.

Results

Both markers were first compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among wild C. canephora genotypes. As expected, SSR markers were found to have a higher genetic distance detection capacity than RFLP. Nevertheless, similarity matrices showed significant correlations when Mantel's test was carried out (r = 0.66, p < 0.0001). Finally, both markers were equally effective for group discrimination and phylogenetic studies, but SSR markers tended to outperform RFLP markers in discriminating the source of an individual among diversity groups and in putative hybrid detection. Five well defined genetic groups, one in the Upper Guinean forests, the four others in the Lower Guinean forests, were identified, corresponding to geographical patterning in the individuals.

Conclusion

Our data suggested that the Dahomey Gap, a biogeographical barrier, played a role in wild C. canephora differentiation. Climatic variations during the Pleistocene and/or Holocene probably caused the subgroup differentiation in the Congolese zone through the presence of a mosaic of putative refugia. Recent hybridization between C. canephora diversity groups, both for spontaneous individuals and cultivars, was further characterised according to their geographic dissemination or breeding history as a consequence of human activities.
Identification and characterization of pseudogenes in the rice gene complement
Authors: Thibaud-Nissen Françoise , Ouyang Shu , Buell C Robin
Published: July 2009

Background

The Osa1 Genome Annotation of rice (Oryza sativa L. ssp. japonica cv. Nipponbare) is the product of a semi-automated pipeline that does not explicitly predict pseudogenes. As such, it is likely to mis-annotate pseudogenes as functional genes. A total of 22,033 gene models within the Osa1 Release 5 were investigated as potential pseudogenes as these genes exhibit at least one feature potentially indicative of pseudogenes: lack of transcript support, short coding region, long untranslated region, or, for genes residing within a segmentally duplicated region, lack of a paralog or significantly shorter corresponding paralog.

Results

A total of 1,439 pseudogenes, identified among genes with pseudogene features, were characterized by similarity to fully-supported gene models and the presence of frameshifts or premature translational stop codons. Significant difference in the length of duplicated genes within segmentally-duplicated regions was the optimal indicator of pseudogenization. Among the 816 pseudogenes for which a probable origin could be determined, 75% originated from gene duplication events while 25% were the result of retrotransposition events. A total of 12% of the pseudogenes were expressed. Finally, F-box proteins, BTB/POZ proteins, terpene synthases, chalcone synthases and cytochrome P450 protein families were found to harbor large numbers of pseudogenes.

Conclusion

These pseudogenes still have a detectable open reading frame and are thus distinct from pseudogenes detected within intergenic regions which typically lack definable open reading frames. Families containing the highest number of pseudogenes are fast-evolving families involved in ubiquitination and secondary metabolism.
The formation of acetylcholine receptor clusters visualized with quantum dots
Authors: Geng Lin , Zhang Hailong L , Peng H Benjamin
Published: July 2009

Background

Motor innervation of skeletal muscle leads to the assembly of acetylcholine receptor (AChR) clusters in the postsynaptic membrane at the vertebrate neuromuscular junction (NMJ). Synaptic AChR aggregation, according to the diffusion-mediated trapping hypothesis, involves the establishment of a postsynaptic scaffold that "traps" freely diffusing receptors into forming high-density clusters. Although this hypothesis is widely cited to explain the formation of postsynaptic AChR clusters, direct evidence at molecular level is lacking.

Results

Using quantum dots (QDs) and live cell imaging, we provide new measurements supporting the diffusion-trap hypothesis as applied to AChR cluster formation. Consistent with published works, experiments on cultured Xenopus myotomal muscle cells revealed that AChRs at clusters that formed spontaneously (pre-patterned clusters, also called hot spots) and at those induced by nerve-innervation or by growth factor-coated latex beads were very stable whereas diffuse receptors outside these regions were mobile. Moreover, despite the restriction of AChR movement at sites of synaptogenic stimulation, individual receptors away from these domains continued to exhibit free diffusion, indicating that AChR clustering at NMJ does not involve an active attraction of receptors but is passive and diffusion-driven.

Conclusion

Single-molecular tracking using QDs has provided direct evidence that the clustering of AChRs in muscle cells in response to synaptogenic stimuli is achieved by two distinct cellular processes: the Brownian motion of receptors in the membrane and their trapping and immobilization at the synaptic specialization. This study also provides a clearer picture of the "trap" that it is not a uniformly sticky area but consists of discrete foci at which AChRs are immobilized.
A toolbox for the fast information analysis of multiple-site LFP, EEG and spike train recordings
Authors: Magri Cesare , Whittingstall Kevin , Singh Vanessa , Logothetis Nikos K , Panzeri Stefano
Published: July 2009

Background

Information theory is an increasingly popular framework for studying how the brain encodes sensory information. Despite its widespread use for the analysis of spike trains of single neurons and of small neural populations, its application to the analysis of other types of neurophysiological signals (EEGs, LFPs, BOLD) has remained relatively limited so far. This is due to the limited-sampling bias which affects calculation of information, to the complexity of the techniques to eliminate the bias, and to the lack of publicly available fast routines for the information analysis of multi-dimensional responses.

Results

Here we introduce a new C- and Matlab-based information theoretic toolbox, specifically developed for neuroscience data. This toolbox implements a novel computationally-optimized algorithm for estimating many of the main information theoretic quantities and bias correction techniques used in neuroscience applications. We illustrate and test the toolbox in several ways. First, we verify that these algorithms provide accurate and unbiased estimates of the information carried by analog brain signals (i.e. LFPs, EEGs, or BOLD) even when using limited amounts of experimental data. This test is important since existing algorithms were so far tested primarily on spike trains. Second, we apply the toolbox to the analysis of EEGs recorded from a subject watching natural movies, and we characterize the electrodes locations, frequencies and signal features carrying the most visual information. Third, we explain how the toolbox can be used to break down the information carried by different features of the neural signal into distinct components reflecting different ways in which correlations between parts of the neural signal contribute to coding. We illustrate this breakdown by analyzing LFPs recorded from primary visual cortex during presentation of naturalistic movies.

Conclusion

The new toolbox presented here implements fast and data-robust computations of the most relevant quantities used in information theoretic analysis of neural data. The toolbox can be easily used within Matlab, the environment used by most neuroscience laboratories for the acquisition, preprocessing and plotting of neural data. It can therefore significantly enlarge the domain of application of information theory to neuroscience, and lead to new discoveries about the neural code.
Effectiveness and cost of recruitment strategies for a community-based randomised controlled trial among rainwater drinkers
Authors: Rodrigo Shelly , Sinclair Martha , Cunliffe David , Leder Karin
Published: July 2009

Background

Community-based recruitment is challenging particularly if the sampling frame is not easily defined as in the case of people who drink rainwater. Strategies for contacting participants must be carefully considered to maximise generalisability and minimise bias of the results. This paper assesses the recruitment strategies for a 1-year double-blinded randomised trial on drinking untreated rainwater. The effectiveness of the recruitment strategies and associated costs are described.

Methods

Community recruitment of households from Adelaide, Australia occurred from February to July 2007 using four methods: electoral roll mail-out, approaches to schools and community groups, newspaper advertising, and other media involvement. Word of mouth communication was also assessed.

Results

A total of 810 callers were screened, with 53.5% eligible. Of those who were eligible and sent further information, 76.7% were willing to participate in the study and 75.1% were enrolled. The target for recruitment was 300 households, and this was achieved. The mail-out was the most effective method with respect to number of households randomised, while recruitment via schools had the highest yield (57.3%) and was the most cost effective when considering cost per household randomised (AUD$147.20). Yield and cost effectiveness were lowest for media advertising.

Conclusion

The use of electoral roll mail-out and advertising via schools were effective in reaching households using untreated rainwater for drinking. Employing multiple strategies enabled success in achieving the recruitment target. In countries where electoral roll extracts are available to researchers, this method is likely to have a high yield for recruitment into community-based epidemiological studies.
Characterization of the patterns of drug-resistance mutations in newly diagnosed HIV-1 infected patients naïve to the antiretroviral drugs
Authors: Alteri Claudia , Svicher Valentina , Gori Caterina , D'Arrigo Roberta , Ciccozzi Massimo , Ceccherini-Silberstein Francesca , Selleri Marina , Bardacci Stefano Aviani , Giuliani Massimo , Elia Paola , Scognamiglio Paola , Balzano Roberta , Orchi Nicoletta , Girardi Enrico , Perno Carlo Federico ,   SENDIH Study Group
Published: July 2009

Background

The transmission of HIV-1 drug-resistant strains in drug naive patients may seriously compromise the efficacy of a first-line antiretroviral treatment. To better define this problem, a study in a cohort of newly diagnosed HIV-1 infected individuals has been conducted. This study is aimed to assess the prevalence and the patterns of the mutations recently associated with transmitted drug resistance in the reverse transcriptase (RT) and in protease (PR) of HIV-1.

Methods

Prevalence of transmitted drug resistant strains is determined in 255 newly diagnosed HIV-1 infected patients enrolled in different counselling and testing (CT) centres in Central Italy; the Avidity Index (AI) on the first available serum sample is also used to estimate time since infection. Logistic regression models are used to determine factors associated with infection by drug resistant HIV-1 strains.

Results

The prevalence of HIV-1 strains with at least one major drug resistance mutation is 5.9% (15/255); moreover, 3.9% (10/255) of patients is infected with HIV nucleoside reverse transcriptase inhibitor (NRTI)-resistant viruses, 3.5% (9/255) with HIV non-NRTI-resistant viruses and 0.4% (1/255) with HIV protease inhibitor (PI)-resistant viruses. Most importantly, almost half (60.0%) of patients carries HIV-1 resistant strains with more than one major drug resistance mutation. In addition, patients who had acquired HIV through homosexual intercourses are more likely to harbour a virus with at least one primary resistance mutation (OR 7.7; 95% CI: 1.7–35.0, P = 0.008).

Conclusion

The prevalence of drug resistant HIV-1 strains among newly diagnosed individuals in Central Italy is consistent with the data from other European countries. Nevertheless, the presence of drug-resistance HIV-1 mutations in complex patterns highlights an additional potential risk for public health and strongly supports the extension of wide genotyping to newly diagnosed HIV-1 infected patients.
Absence of association between SERPINE2genetic polymorphisms and chronic obstructive pulmonary disease in Han Chinese: a case-control cohort study
Authors: Zhong Li , Fu Wei-Ping , Sun Chang , Dai Lu-Ming , Zhang Ya-Ping
Published: July 2009

Background

Recent studies have proposed that the serine protease inhibitor E2 (SERPINE2) was a novel susceptibility gene for chronic obstructive pulmonary disease (COPD) in Caucasians. However, this issue still remained controversial. Additional evidences from populations with different environments and/or genetic backgrounds, such as East Asian, would be helpful to elucidate the issue.

Methods

In this study, five proposed causal SNPs in SERPINE2 were genotyped in 327 COPD patients and 349 controls, all of which belonged to the Han population sampled from Southwest China. The frequency of each SNP was compared both individually and in combination between patients and controls. The potential relationship between these SNPs and severity of COPD was also investigated.

Results

Three SNPs (rs3795877, rs6747096, and rs3795879) showed complete linkage disequilibrium (r2 = 1), and the minor allele frequencies were 13.0% and 12.9% in case and control cohorts, respectively, with no significant difference observed (P = 0.96). We also failed to observe any significant correlation between these SNPs and COPD severity (P = 0.67). The other two SNPs (rs7579646 and rs840088) also presented a similar pattern. Moreover, four major haplotypes were observed in our sample but none showed a significant difference between case and control groups (P > 0.1).

Conclusion

Our results failed to obtain the evidence that these SNPs in SERPINE2 contributed to the COPD susceptibility in the Han Chinese population.
Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer
Authors: Jörißen Hannah , Bektas Nuran , Dahl Edgar , Hartmann Arndt , ten Haaf Anette , Di Fiore Stefano , Kiefer Hans , Thess Andreas , Barth Stefan , Klockenbring Torsten
Published: July 2009

Background

RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens.

Methods

We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression.

Results

The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival.

Conclusion

We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human breast tissue at both the mRNA and protein levels.
Low-risk susceptibility alleles in 40 human breast cancer cell lines
Authors: Riaz Muhammad , Elstrodt Fons , Hollestelle Antoinette , Dehghan Abbas , Klijn Jan GM , Schutte Mieke
Published: July 2009

Background

Low-risk breast cancer susceptibility alleles or SNPs confer only modest breast cancer risks ranging from just over 1.0 to1.3 fold. Yet, they are common among most populations and therefore are involved in the development of essentially all breast cancers. The mechanism by which the low-risk SNPs confer breast cancer risks is currently unclear. The breast cancer association consortium BCAC has hypothesized that the low-risk SNPs modulate expression levels of nearby located genes.

Methods

Genotypes of five low-risk SNPs were determined for 40 human breast cancer cell lines, by direct sequencing of PCR-amplified genomic templates. We have analyzed expression of the four genes that are located nearby the low-risk SNPs, by using real-time RT-PCR and Human Exon microarrays.

Results

The SNP genotypes and additional phenotypic data on the breast cancer cell lines are presented. We did not detect any effect of the SNP genotypes on expression levels of the nearby-located genesMAP3K1, FGFR2, TNRC9andLSP1.

Conclusion

The SNP genotypes provide a base line for functional studies in a well-characterized cohort of 40 human breast cancer cell lines. Our expression analyses suggest that a putative disease mechanism through gene expression modulation is not operative in breast cancer cell lines.
Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies
Authors: Edelheit Oded , Hanukoglu Aaron , Hanukoglu Israel
Published: July 2009

Background

In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction.

Results

In initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95°C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na+ Channel (ENaC) subunits. The method has been tested for 1–3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs.

Conclusion

The SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction.
Involving students in real-world research: a pilot study for teaching public health and research skills
Authors: Millar Elinor , Baker Michael G , Howden-Chapman Philippa , Wilson Nick , Dickson Nigel
Published: July 2009

Background

There is some evidence that medical students consider population health issues less important than other domains in the health sciences and attitudes to this field may become more negative as training progresses. A need to improve research skills among medical students has also been suggested. Therefore we piloted an integrative teaching exercise that combined teaching of research skills and public health, with real-world research.

Methods

Third year medical students at the University of Otago (Dunedin, New Zealand) filled in a questionnaire on their housing conditions and health. The students were given the results of the survey to discuss in a subsequent class. Student response to this teaching exercise was assessed using a Course Evaluation Questionnaire.

Results

Of the 210 students in the class, 136 completed the Course Evaluation Questionnaire (65%). A majority of those who responded (77%) greatly supported or supported the use of the survey and seminar discussion for future third year classes. Most (70%) thought that the session had made them more aware and concerned about societal problems, and 72% felt that they now had an improved understanding of the environmental determinants of health. Students liked the relevance and interaction of the session, but thought it could be improved by the inclusion of small group discussion. The findings of the students' housing and health were considered by the tutors to be of sufficient value to submit to a scientific journal and are now contributing to community action to improve student housing in the city.

Conclusion

In this pilot study it was feasible to integrate medical student teaching with real-world research. A large majority of the students responded favourably to the teaching exercise and this was generally successful in raising the profile of public health and research. This approach to integrated teaching/research should be considered further in health sciences training and continue to be evaluated and refined.
Temporal stability of naturally acquired immunity to Merozoite Surface Protein-1 in Kenyan Adults
Authors: Dent Arlene E , Chelimo Kiprotich , Sumba Peter O , Spring Michele D , Crabb Brendan S , Moormann Ann M , Tisch Daniel J , Kazura James W
Published: July 2009

Background

Naturally acquired immunity to blood-stage Plasmodium falciparum infection develops with age and after repeated infections. In order to identify immune surrogates that can inform vaccine trials conducted in malaria endemic populations and to better understand the basis of naturally acquired immunity it is important to appreciate the temporal stability of cellular and humoral immune responses to malaria antigens.

Methods

Blood samples from 16 adults living in a malaria holoendemic region of western Kenya were obtained at six time points over the course of 9 months. T cell immunity to the 42 kDa C-terminal fragment of Merozoite Surface Protein-1 (MSP-142) was determined by IFN-γ ELISPOT. Antibodies to the 42 kDa and 19 kDa C-terminal fragments of MSP-1 were determined by serology and by functional assays that measure MSP-119 invasion inhibition antibodies (IIA) to the E-TSR (3D7) allele and growth inhibitory activity (GIA). The haplotype of MSP-119 alleles circulating in the population was determined by PCR. The kappa test of agreement was used to determine stability of immunity over the specified time intervals of 3 weeks, 6 weeks, 6 months, and 9 months.

Results

MSP-1 IgG antibodies determined by serology were most consistent over time, followed by MSP-1 specific T cell IFN-γ responses and GIA. MSP-119 IIA showed the least stability over time. However, the level of MSP-119 specific IIA correlated with relatively higher rainfall and higher prevalence of P. falciparum infection with the MSP-119 E-TSR haplotype.

Conclusion

Variation in the stability of cellular and humoral immune responses to P. falciparum blood stage antigens needs to be considered when interpreting the significance of these measurements as immune endpoints in residents of malaria endemic regions.
Altered proteolytic events in experimental autoimmune encephalomyelitis discovered by iTRAQ shotgun proteomics analysis of spinal cord
Authors: Jain Mohit Raja , Bian Shengjie , Liu Tong , Hu Jun , Elkabes Stella , Li Hong
Published: July 2009

Background

Abnormal activation of protease activities during experimental autoimmune encephalomyelitis (EAE) in rats, a rodent model of multiple sclerosis, have been implicated in either the direct destruction of myelin components or the intracellular signal transduction pathways that lead to lymphocyte infiltration, oligodendrocyte destruction, neuronal dysfunctions and axonal degeneration. The identification of changes in regulated proteolytic events during EAE is crucial for uncovering activated proteases that may underline the pathological features such as inflammation and demyelination. We searched for either non-tryptic or semi-tryptic peptides from a previous shotgun proteomics study using isobaric tags for relative and absolute quantification (iTRAQ) to compare the proteomes of normal and EAE rat lumbar spinal cords.

Results

We discovered that several proteins, such as α1-macroglobulin, a protease inhibitor, α1B-glycoprotein, β2-microglobulin, neurofilament light polypeptide and sulfated glycoprotein 1 had non-tryptic peptide iTRAQ ratios that were substantially different from the overall protein iTRAQ ratios, suggesting that such peptides may be markers for the proteolytic products generated by the protease(s) altered during EAE. Indeed, subsequent Western blotting confirmed the dysregulation of specific protein cleavages in EAE tissues. Additional proteolytic changes in α2-macroglobulin, another protease inhibitor similar to α1-macroglobulin was also observed.

Conclusion

The results from this study revealed changes among both neuronal protein processing and endogenous proteolysis modulators in EAE animals. This information may provide a rationale for protease inhibitor-based therapeutic interventions for multiple sclerosis.
Molecular and functional characterization of voltage-gated sodium channels in human sperm
Authors: Pinto Francisco M , Ravina Cristina G , Fernández-Sánchez Manuel , Gallardo-Castro Manuel , Cejudo-Román Antonio , Candenas Luz
Published: July 2009

Background

We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility.

Methods

Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2.

Results

The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels.

Conclusion

This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.
Hypoglycemic and beta cell protective effects of andrographolide analogue for diabetes treatment
Authors: Zhang Zaijun , Jiang Jie , Yu Pei , Zeng Xiangping , Larrick James W , Wang Yuqiang
Published: July 2009

Background

While all anti-diabetic agents can decrease blood glucose level directly or indirectly, few are able to protect and preserve both pancreatic beta cell mass and their insulin-secreting functions. Thus, there is an urgent need to find an agent or combination of agents that can lower blood glucose and preserve pancreatic beta cells at the same time. Herein, we report a dual-functional andrographolide-lipoic acid conjugate (AL-1). The anti-diabetic and beta cell protective activities of this novel andrographolide-lipoic acid conjugate were investigated.

Methods

In alloxan-treated mice (a model of type 1 diabetes), drugs were administered orally once daily for 6 days post-alloxan treatment. Fasting blood glucose and serum insulin were determined. Pathologic and immunohistochemical analysis of pancreatic islets were performed. Translocation of glucose transporter subtype 4 in soleus muscle was detected by western blot. In RIN-m cellsin vitro, the effect of AL-1 on H2O2-induced damage and reactive oxidative species production stimulated by high glucose and glibenclamide were measured. Inhibition of nuclear factor kappa B (NF-κB) activation induced by IL-1β and IFN-γ was investigated.

Results

In alloxan-induced diabetic mouse model, AL-1 lowered blood glucose, increased insulin and prevented loss of beta cells and their dysfunction, stimulated glucose transport protein subtype 4 (GLUT4) membrane translocation in soleus muscles. Pretreatment of RIN-m cells with AL-1 prevented H2O2-induced cellular damage, quenched glucose and glibenclamide-stimulated reactive oxidative species production, and inhibited cytokine-stimulated NF-κB activation.

Conclusion

We have demonstrated that AL-1 had both hypoglycemic and beta cell protective effects which translated into antioxidant and NF-κB inhibitory activity. AL-1 is a potential new anti-diabetic agent.
Proviral integrations and expression of endogenous Avian leucosis virus during long term selection for high and low body weight in two chicken lines
Authors: Ka Sojeong , Kerje Susanne , Bornold Lina , Liljegren Ulrika , Siegel Paul B , Andersson Leif , Hallböök Finn
Published: July 2009

Background

Long-term selection (> 45 generations) for low or high juvenile body weight from a common founder population of White Plymouth Rock chickens has generated two extremely divergent lines, the LWS and HWS lines. In addition to a > 9-fold difference between lines for the selected trait, large behavioural and metabolic differences between the two lines evolved during the course of the selection. We recently compared gene expression in brain tissue from birds representing these lines using a global cDNA array analysis and the results showed multiple but small expression differences in protein coding genes. The main differentially expressed transcripts were endogenous retroviral sequences identified as avian leucosis virus subgroup-E (ALVE).

Results

In this work we confirm the differential ALVE expression and analysed expression and number of proviral integrations in the two parental lines as well as in F9 individuals from an advanced intercross of the lines. Correlation analysis between expression, proviral integrations and body weight showed that high ALVE levels in the LWS line were inherited and that more ALVE integrations were detected in LWS than HWS birds.

Conclusion

We conclude that only a few of the integrations contribute to the high expression levels seen in the LWS line and that high ALVE expression was significantly correlated with lower body weights for the females but not males. The conserved correlation between high expression and low body weight in females after 9 generations of intercrosses, indicated that ALVE loci conferring high expression directly affects growth or are very closely linked to loci regulating growth.
Spatial distribution of the active surveillance of sheep scrapie in Great Britain: an exploratory analysis
Authors: Birch Colin PD , Chikukwa Ambrose C , Hyder Kieran , Del Rio Vilas Victor J
Published: July 2009

Background

This paper explores the spatial distribution of sampling within the active surveillance of sheep scrapie in Great Britain. We investigated the geographic distribution of the birth holdings of sheep sampled for scrapie during 2002 – 2005, including samples taken in abattoir surveys (c. 83,100) and from sheep that died in the field ("fallen stock", c. 14,600). We mapped the birth holdings by county and calculated the sampling rate, defined as the proportion of the holdings in each county sampled by the surveys. The Moran index was used to estimate the global spatial autocorrelation across Great Britain. The contributions of each county to the global Moran index were analysed by a local indicator of spatial autocorrelation (LISA).

Results

The sampling rate differed among counties in both surveys, which affected the distribution of detected cases of scrapie. Within each survey, the county sampling rates in different years were positively correlated during 2002–2005, with the abattoir survey being more strongly autocorrelated through time than the fallen stock survey. In the abattoir survey, spatial indices indicated that sampling rates in neighbouring counties tended to be similar, with few significant contrasts. Sampling rates were strongly correlated with sheep density, being highest in Wales, Southwest England and Northern England. This relationship with sheep density accounted for over 80% of the variation in sampling rate among counties. In the fallen stock survey, sampling rates in neighbouring counties tended to be different, with more statistically significant contrasts. The fallen stock survey also included a larger proportion of holdings providing many samples.

Conclusion

Sampling will continue to be uneven unless action is taken to make it more uniform, if more uniform sampling becomes a target. Alternatively, analyses of scrapie occurrence in these datasets can take account of the distribution of sampling. Combining the surveys only partially reduces uneven sampling. Adjusting the distribution of sampling between abattoirs to reduce the bias in favour of regions with high sheep densities could probably achieve more even sampling. However, any adjustment of sampling should take account of the current understanding of the distribution of scrapie cases, which will be improved by further analysis of this dataset.
Deterministic mathematical models of the cAMP pathway in Saccharomyces cerevisiae
Authors: Williamson Thomas , Schwartz Jean-Marc , Kell Douglas B , Stateva Lubomira
Published: July 2009

Background

Cyclic adenosine monophosphate (cAMP) has a key signaling role in all eukaryotic organisms. In Saccharomyces cerevisiae, it is the second messenger in the Ras/PKA pathway which regulates nutrient sensing, stress responses, growth, cell cycle progression, morphogenesis, and cell wall biosynthesis. A stochastic model of the pathway has been reported.

Results

We have created deterministic mathematical models of the PKA module of the pathway, as well as the complete cAMP pathway. First, a simplified conceptual model was created which reproduced the dynamics of changes in cAMP levels in response to glucose addition in wild-type as well as cAMP phosphodiesterase deletion mutants. This model was used to investigate the role of the regulatory Krh proteins that had not been included previously. The Krh-containing conceptual model reproduced very well the experimental evidence supporting the role of Krh as a direct inhibitor of PKA. These results were used to develop the Complete cAMP Model. Upon simulation it illustrated several important features of the yeast cAMP pathway: Pde1p is more important than is Pde2p for controlling the cAMP levels following glucose pulses; the proportion of active PKA is not directly proportional to the cAMP level, allowing PKA to exert negative feedback; negative feedback mechanisms include activating Pde1p and deactivating Ras2 via phosphorylation of Cdc25. The Complete cAMP model is easier to simulate, and although significantly simpler than the existing stochastic one, it recreates cAMP levels and patterns of changes in cAMP levels observed experimentally in vivo in response to glucose addition in wild-type as well as representative mutant strains such as pde1Δ, pde2Δ, cyr1Δ, and others. The complete model is made available in SBML format.

Conclusion

We suggest that the lower number of reactions and parameters makes these models suitable for integrating them with models of metabolism or of the cell cycle in S. cerevisiae. Similar models could be also useful for studies in the human pathogen Candida albicans as well as other less well-characterized fungal species.
Using microarrays to identify positional candidate genes for QTL: the case study of ACTH response in pigs
Authors: Jouffe Vincent , Rowe Suzanne , Liaubet Laurence , Buitenhuis Bart , Hornshøj Henrik , SanCristobal Magali , Mormède Pierre , de Koning DJ
Published: July 2009

Background

Microarray studies can supplement QTL studies by suggesting potential candidate genes in the QTL regions, which by themselves are too large to provide a limited selection of candidate genes. Here we provide a case study where we explore ways to integrate QTL data and microarray data for the pig, which has only a partial genome sequence. We outline various procedures to localize differentially expressed genes on the pig genome and link this with information on published QTL. The starting point is a set of 237 differentially expressed cDNA clones in adrenal tissue from two pig breeds, before and after treatment with adrenocorticotropic hormone (ACTH).

Results

Different approaches to localize the differentially expressed (DE) genes to the pig genome showed different levels of success and a clear lack of concordance for some genes between the various approaches. For a focused analysis on 12 genes, overlapping QTL from the public domain were presented. Also, differentially expressed genes underlying QTL for ACTH response were described. Using the latest version of the draft sequence, the differentially expressed genes were mapped to the pig genome. This enabled co-location of DE genes and previously studied QTL regions, but the draft genome sequence is still incomplete and will contain many errors. A further step to explore links between DE genes and QTL at the pathway level was largely unsuccessful due to the lack of annotation of the pig genome. This could be improved by further comparative mapping analyses but this would be time consuming.

Conclusion

This paper provides a case study for the integration of QTL data and microarray data for a species with limited genome sequence information and annotation. The results illustrate the challenges that must be addressed but also provide a roadmap for future work that is applicable to other non-model species.
sigReannot: an oligo-set re-annotation pipeline based on similarities with the Ensembl transcripts and Unigene clusters
Authors: Casel Pierrot , Moreews François , Lagarrigue Sandrine , Klopp Christophe
Published: July 2009

Background

Microarray is a powerful technology enabling to monitor tens of thousands of genes in a single experiment. Most microarrays are now using oligo-sets. The design of the oligo-nucleotides is time consuming and error prone. Genome wide microarray oligo-sets are designed using as large a set of transcripts as possible in order to monitor as many genes as possible. Depending on the genome sequencing state and on the assembly state the knowledge of the existing transcripts can be very different. This knowledge evolves with the different genome builds and gene builds. Once the design is done the microarrays are often used for several years. The biologists working in EADGENE expressed the need of up-to-dated annotation files for the oligo-sets they share including information about the orthologous genes of model species, the Gene Ontology, the corresponding pathways and the chromosomal location.

Results

The results of SigReannot on a chicken micro-array used in the EADGENE project compared to the initial annotations show that 23% of the oligo-nucleotide gene annotations were not confirmed, 2% were modified and 1% were added. The interest of this up-to-date annotation procedure is demonstrated through the analysis of real data previously published.

Conclusion

SigReannot uses the oligo-nucleotide design procedure criteria to validate the probe-gene link and the Ensembl transcripts as reference for annotation. It therefore produces a high quality annotation based on reference gene sets.
OligoRAP – an Oligo Re-Annotation Pipeline to improve annotation and estimate target specificity
Authors: Neerincx Pieter BT , Rauwerda Han , Nie Haisheng , Groenen Martien AM , Breit Timo M , Leunissen Jack AM
Published: July 2009

Background

High throughput gene expression studies using oligonucleotide microarrays depend on the specificity of each oligonucleotide (oligo or probe) for its target gene. However, target specific probes can only be designed when a reference genome of the species at hand were completely sequenced, when this genome were completely annotated and when the genetic variation of the sampled individuals were completely known. Unfortunately there is not a single species for which such a complete data set is available. Therefore, it is important that probe annotation can be updated frequently for optimal interpretation of microarray experiments.

Results

In this paper we present OligoRAP, a pipeline to automatically update the annotation of oligo libraries and estimate oligo target specificity. OligoRAP uses a reference genome assembly with Ensembl and Entrez Gene annotation supplemented with a set of unmapped transcripts derived from RefSeq and UniGene to handle assembly gaps. OligoRAP produces alignments of each oligo with the reference assembly as well as with unmapped transcripts. These alignments are re-mapped to the annotation sources, which results in a concise, as complete as possible and up-to-date annotation of the oligo library. The building blocks of this pipeline are BioMoby web services creating a highly modular and distributed system with a robust, remote programmatic interface.

OligoRAP was used to update the annotation for a subset of 791 oligos from the ARK-Genomics 20 K chicken array, which were selected as starting material for the oligo annotation session of the EADGENE/SABRE Post-analysis workshop. Based on the updated annotation about one third of these oligos is problematic with regard to target specificity. In addition, the accession numbers or ids the oligos were originally designed for no longer exist in the updated annotation for almost half of the oligos.

Conclusion

As microarrays are designed on incomplete data, it is important to update probe annotation and check target specificity regularly. OligoRAP provides both and due to its design based on BioMoby web services it can easily be embedded as an oligo annotation engine in customised applications for microarray data analysis. The dramatic difference in updated annotation and target specificity for the ARK-Genomics 20 K chicken array as compared to the original data emphasises the need for regular updates.
Gene set analysis methods applied to chicken microarray expression data
Authors: Skarman Axel , Jiang Li , Hornshøj Henrik , Buitenhuis Bart , Hedegaard Jakob , Conley Lene N , Sørensen Peter
Published: July 2009

Background

Gene set analysis is considered to be a way of improving our biological interpretation of the observed expression patterns. This paper describes different methods applied to analyse expression data from a chicken DNA microarray dataset.

Results

Applying different gene set analyses to the chicken expression data led to different ranking of the Gene Ontology terms tested. A method for prediction of possible annotations was applied.

Conclusion

Biological interpretation based on gene set analyses dependent on the statistical method used. Methods for predicting the possible annotations for genes with unknown function from the expression data at hand could be useful, but our results indicate that careful validation of the predictions is needed.