bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

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

AbstractBackgroundWhile 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.MethodsIn 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.ResultsIn 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.ConclusionWe 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

Abstract

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

AbstractBackgroundThis 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).ResultsThe 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.ConclusionSampling 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

Abstract

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

Abstract

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

Abstract

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.