bluuebunny/crossref_metadata_2025 huggingface dataset:
Social Comparison within Self-help Groups
Authors: Hodges Lucy , Dibb Bridget
Published: May 2010

This study used a qualitative approach to examine the experiences of parents participating as members of the Duchenne Family Support Group (DFSG), with a focus on whether social comparison occurred within this group. Eight parents were interviewed about their experiences of being a member. Thematic analysis was used to identify themes inductively. The study uncovered a wide range of social comparisons, including upward and downward comparisons on illness and coping dimensions, with both positive and negative affects. The results highlighted the complexity of socially comparing within the context of chronic illness, particularly in an illness in which deterioration is inevitable.
On the Costs of Self-interested Economic Behavior
Authors: Dunn Elizabeth W. , Ashton-James Claire E. , Hanson Margaret D. , Aknin Lara B.
Published: May 2010

The present study examined how financial decisions ‘get under the skin’. Participants played an economic game in which they could donate some of their payment to another student. Affect was measured afterward and salivary cortisol was measured before and afterward. Participants who kept more money for themselves reported less positive affect, more negative affect, and more shame. Shame predicted higher levels of post-game cortisol, controlling for pre-game cortisol; stingy economic behavior therefore produced a significant indirect effect on cortisol via shame. Thus, shame and cortisol represent plausible emotional and biological pathways linking everyday decisions with downstream consequences for health.
Residential Status Moderates the Association between Gender and Risky Sexual Behavior
Authors: Hittner James B. , Kryzanowski Jennifer J.
Published: May 2010

College students’ academic performance, residential status, class rank, and gender were assessed as predictors of risky sexual behavior. All predictors were examined under two different substance use contexts, that of being ‘drunk or high’ and ‘not drunk or high’. Results indicated that gender moderated the association between residential status and risky sex, such that males living on-campus engaged in more frequent casual sex than males living off-campus. The casual sex frequency for females did not vary as a function of residential status. The importance of studying the link between college-based environmental variables and risky sexual behavior was emphasized.
Theoretical Consoles: Concepts for Gadget Analysis
Authors: Verhoeff Nanna
Published: May 2010

Theoretical objects are things that compel us to propose, interrogate, and theorize. They counter the influence of approaches that try to define, position, and fix. The mobile, handheld game console offers us a specific kind of theoretical object. A hybrid screen device that encompasses multiple interfaces, it raises questions about the specificity of the screen gadget as object, and about the entanglement of technologies, applications, and practices. Through an analysis of the Nintendo DS game console I argue that such gadgets are best understood as theoretical consoles: objects that raises theoretical questions, precisely, about their hybrid status.
In Conversation
Authors: Stallabrass Julian , Gilbertson Ashley
Published: May 2010

The photojournalist, Ashley Gilbertson, and the curator of the 2008 Brighton Photo Biennial, Julian Stallabrass, discuss the practice of war photography with particular reference to the Iraq War and the Pentagon’s embedding system. They explore the relations of photojournalists with soldiers and the public, the military’s efforts at public relations, the resistance’s relative lack of interest in the Western media, the restrictions and possibilities offered by embedding, the ethics of photojournalistic practice, and the marked fall-off in coverage of the Iraq War.
Examination of an Administrator-Read Vocabulary-Matching Measure as an Indicator of Science Achievement
Authors: Borsuk Ellen R.
Published: May 2010

Research and development of measures to document ongoing learning within the content areas are in the beginning stages. As such, the current study adds to efforts in the development of the vocabulary-matching measure. Using a modified format of the previously studied vocabulary-matching measure, 63 middle school students completed alternate forms of an administrator-read science measure weekly across 24 data collection periods. Results of hierarchical linear modeling analysis revealed a significant mean growth rate across time (0.26 items per week), significant individual variation in learning rates, and growth rate differences that were significantly predicted by student disability status. General education students had a higher rate of growth than those with disabilities included for science. Results are analyzed and limitations as well as directions for future research are discussed. It is concluded that current findings lend additional validity support to the interpretation of vocabulary-matching scores as indices of learning in the content areas.
Review: Targeting the Hedgehog pathway in cancer
Authors: Gupta Sachin , Takebe Naoko , LoRusso Patricia
Published: May 2010

The Hedgehog (Hh) pathway is a major regulator of many fundamental processes in vertebrate embryonic development including stem cell maintenance, cell differentiation, tissue polarity and cell proliferation. Constitutive activation of the Hh pathway leading to tumorigenesis is seen in basal cell carcinomas and medulloblastoma. A variety of other human cancers, including brain, gastrointestinal, lung, breast and prostate cancers, also demonstrate inappropriate activation of this pathway. Paracrine Hh signaling from the tumor to the surrounding stroma was recently shown to promote tumorigenesis. This pathway has also been shown to regulate proliferation of cancer stem cells and to increase tumor invasiveness. Targeted inhibition of Hh signaling may be effective in the treatment and prevention of many types of human cancers. The discovery and synthesis of specific Hh pathway inhibitors have significant clinical implications in novel cancer therapeutics. Several synthetic Hh antagonists are now available, several of which are undergoing clinical evaluation. The orally available compound, GDC-0449, is the farthest along in clinical development. Initial clinical trials in basal cell carcinoma and treatment of select patients with medulloblastoma have shown good efficacy and safety. We review the molecular basis of Hh signaling, the current understanding of pathway activation in different types of human cancers and we discuss the clinical development of Hh pathway inhibitors in human cancer therapy.
A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells
Authors: Zhou Sheng , Mody Disha , DeRavin Suk See , Hauer Julia , Lu Taihe , Ma Zhijun , Hacein-Bey Abina Salima , Gray John T. , Greene Michael R. , Cavazzana-Calvo Marina , Malech Harry L. , Sorrentino Brian P.
Published: May 2010

To develop safer and more effective vectors for gene therapy of X-linked severe combined immunodeficiency (SCID-X1), we have evaluated new self-inactivating lentiviral vectors based on the HIV virus. The CL20i4-hγc-Revgen vector contains the entire human common γ chain (γc) genomic sequence driven by the γc promoter. The CL20i4-EF1α-hγcOPT vector uses a promoter fragment from the eukaryotic elongation factor alpha (EF1α) gene to express a codon-optimized human γc cDNA. Both vectors contain a 400-bp insulator fragment from the chicken β-globin locus within the self-inactivating long-terminal repeat. Transduction of bone marrow cells using either of these vectors restored T, B, and natural killer lymphocyte development and function in a mouse SCID-X1 transplantation model. Transduction of human CD34+ bone marrow cells from SCID-X1 patients with either vector restored T-cell development in an in vitro assay. In safety studies using a Jurkat LMO2 activation assay, only the CL20i4-EF1α-hγcOPT vector lacked the ability to transactivate LMO2 protein expression, whereas the CL20i4-hγc-Revgen vector significantly activated LMO2 protein expression. In addition, the CL20i4-EF1α-hγcOPT vector has not caused any tumors in transplanted mice. We conclude that the CL20i4-EF1α-hγcOPT vector may be suitable for testing in a clinical trial based on these preclinical demonstrations of efficacy and safety.
Critical role of Src-Syk-PLCγ2 signaling in megakaryocyte migration and thrombopoiesis
Authors: Mazharian Alexandra , Thomas Steve G. , Dhanjal Tarvinder S. , Buckley Christopher D. , Watson Steve P.
Published: May 2010

Migration of megakaryocytes (MKs) from the proliferative osteoblastic niche to the capillary-rich vascular niche is essential for proplatelet formation and platelet release. In this study, we explore the role of surface glycoprotein receptors and signaling proteins in regulating MK migration and platelet recovery after immune-induced thrombocytopenia. We show that spreading and migration of mouse primary bone marrow–derived MKs on a fibronectin matrix are abolished by the Src family kinases inhibitor PP1, the Syk kinase inhibitor R406 and the integrin αIIbβ3 antagonist lotrafiban. We also demonstrate that these responses are inhibited in primary phospholipase C γ2 (PLCγ2)–deficient MKs. Conversely, MK spreading and migration were unaltered in the absence of the collagen receptor, the glycoprotein VI–FcRγ-chain complex. We previously reported a correlation between a defect in MK migration and platelet recovery in the absence of platelet endothelial cell adhesion molecule-1 and the tyrosine phosphatase CD148. This correlation also holds for mice deficient in PLCγ2. This study identifies a model in which integrin signaling via Src family kinases and Syk kinase to PLCγ2 is required for MK spreading, migration, and platelet formation.
Trypsin-induced proteome alteration during cell subculture in mammalian cells
Authors: Huang Hsiang-Ling , Hsing Hsiang-Wei , Lai Tzu-Chia , Chen Yi-Wen , Lee Tian-Ren , Chan Hsin-Tsu , Lyu Ping-Chiang , Wu Chieh-Lin , Lu Ying-Chieh , Lin Szu-Ting , Lin Cheng-Wen , Lai Chih-Ho , Chang Hao-Teng , Chou Hsiu-Chuan , Chan Hong-Lin
Published: May 2010

Background

It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.

Methods

In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.

Results

36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.

Conclusions

In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.
A database and API for variation, dense genotyping and resequencing data
Authors: Rios Daniel , McLaren William M , Chen Yuan , Birney Ewan , Stabenau Arne , Flicek Paul , Cunningham Fiona
Published: May 2010

Background

Advances in sequencing and genotyping technologies are leading to the widespread availability of multi-species variation data, dense genotype data and large-scale resequencing projects. The 1000 Genomes Project and similar efforts in other species are challenging the methods previously used for storage and manipulation of such data necessitating the redesign of existing genome-wide bioinformatics resources.

Results

Ensembl has created a database and software library to support data storage, analysis and access to the existing and emerging variation data from large mammalian and vertebrate genomes. These tools scale to thousands of individual genome sequences and are integrated into the Ensembl infrastructure for genome annotation and visualisation. The database and software system is easily expanded to integrate both public and non-public data sources in the context of an Ensembl software installation and is already being used outside of the Ensembl project in a number of database and application environments.

Conclusions

Ensembl's powerful, flexible and open source infrastructure for the management of variation, genotyping and resequencing data is freely available at http://www.ensembl.org.
eHive: An Artificial Intelligence workflow system for genomic analysis
Authors: Severin Jessica , Beal Kathryn , Vilella Albert J , Fitzgerald Stephen , Schuster Michael , Gordon Leo , Ureta-Vidal Abel , Flicek Paul , Herrero Javier
Published: May 2010

Background

The Ensembl project produces updates to its comparative genomics resources with each of its several releases per year. During each release cycle approximately two weeks are allocated to generate all the genomic alignments and the protein homology predictions. The number of calculations required for this task grows approximately quadratically with the number of species. We currently support 50 species in Ensembl and we expect the number to continue to grow in the future.

Results

We present eHive, a new fault tolerant distributed processing system initially designed to support comparative genomic analysis, based on blackboard systems, network distributed autonomous agents, dataflow graphs and block-branch diagrams. In the eHive system a MySQL database serves as the central blackboard and the autonomous agent, a Perl script, queries the system and runs jobs as required. The system allows us to define dataflow and branching rules to suit all our production pipelines. We describe the implementation of three pipelines: (1) pairwise whole genome alignments, (2) multiple whole genome alignments and (3) gene trees with protein homology inference. Finally, we show the efficiency of the system in real case scenarios.

Conclusions

eHive allows us to produce computationally demanding results in a reliable and efficient way with minimal supervision and high throughput. Further documentation is available at:http://www.ensembl.org/info/docs/eHive/.
Error margin analysis for feature gene extraction
Authors: Chow Chi Kin , Zhu Hai Long , Lacy Jessica , Kuo Winston P
Published: May 2010

Background

Feature gene extraction is a fundamental issue in microarray-based biomarker discovery. It is normally treated as an optimization problem of finding the best predictive feature genes that can effectively and stably discriminate distinct types of disease conditions, e.g. tumors and normals. Since gene microarray data normally involves thousands of genes at, tens or hundreds of samples, the gene extraction process may fall into local optimums if the gene set is optimized according to the maximization of classification accuracy of the classifier built from it.

Results

In this paper, we propose a novel gene extraction method of error margin analysis to optimize the feature genes. The proposed algorithm has been tested upon one synthetic dataset and two real microarray datasets. Meanwhile, it has been compared with five existing gene extraction algorithms on each dataset. On the synthetic dataset, the results show that the feature set extracted by our algorithm is the closest to the actual gene set. For the two real datasets, our algorithm is superior in terms of balancing the size and the validation accuracy of the resultant gene set when comparing to other algorithms.

Conclusion

Because of its distinct features, error margin analysis method can stably extract the relevant feature genes from microarray data for high-performance classification.
The long noncoding RNA RNCR2 directs mouse retinal cell specification
Authors: Rapicavoli Nicole A , Poth Erin M , Blackshaw Seth
Published: May 2010

Background

Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs) are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation.

Results

We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation.

Conclusion

These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.
Viral quasispecies profiles as the result of the interplay of competition and cooperation
Authors: Arbiza Juan , Mirazo Santiago , Fort Hugo
Published: May 2010

Background

Viral quasispecies can be regarded as a swarm of genetically related mutants. A common approach employed to describe viral quasispecies is by means of the quasispecies equation (QE). However, a main criticism of QE is its lack of frequency-dependent selection. This can be overcome by an alternative formulation for the evolutionary dynamics: the replicator-mutator equation (RME). In turn, a problem with the RME is how to quantify the interaction coefficients between viral variants. Here, this is addressed by adopting an ecological perspective and resorting to the niche theory of competing communities, which assumes that the utilization of resources primarily determines ecological segregation between competing individuals (the different viral variants that constitute the quasispecies). This provides a theoretical framework to estimate quantitatively the fitness landscape.

Results

Using this novel combination of RME plus the ecological concept of niche overlapping for describing a quasispecies we explore the population distributions of viral variants that emerge, as well as the corresponding dynamics. We observe that the population distribution requires very long transients both to A) reach equilibrium and B) to show a clear dominating master sequence. Based on different independent and recent experimental evidence, we find that when some cooperation or facilitation between variants is included in appropriate doses we can solve both A) and B). We show that a useful quantity to calibrate the degree of cooperation is the Shannon entropy.

Conclusions

In order to get a typical quasispecies profile, at least within the considered mathematical approach, it seems that pure competition is not enough. Some dose of cooperation among viral variants is needed. This has several biological implications that might contribute to shed light on the mechanisms operating in quasispecies dynamics and to understand the quasispecies as a whole entity.
The trans-Saharan slave trade - clues from interpolation analyses and high-resolution characterization of mitochondrial DNA lineages
Authors: Harich Nourdin , Costa Marta D , Fernandes Verónica , Kandil Mostafa , Pereira Joana B , Silva Nuno M , Pereira Luísa
Published: May 2010

Background

A proportion of 1/4 to 1/2 of North African female pool is made of typical sub-Saharan lineages, in higher frequencies as geographic proximity to sub-Saharan Africa increases. The Sahara was a strong geographical barrier against gene flow, at least since 5,000 years ago, when desertification affected a larger region, but the Arab trans-Saharan slave trade could have facilitate enormously this migration of lineages. Till now, the genetic consequences of these forced trans-Saharan movements of people have not been ascertained.

Results

The distribution of the main L haplogroups in North Africa clearly reflects the known trans-Saharan slave routes: West is dominated by L1b, L2b, L2c, L2d, L3b and L3d; the Center by L3e and some L3f and L3w; the East by L0a, L3h, L3i, L3x and, in common with the Center, L3f and L3w; while, L2a is almost everywhere. Ages for the haplogroups observed in both sides of the Saharan desert testify the recent origin (holocenic) of these haplogroups in sub-Saharan Africa, claiming a recent introduction in North Africa, further strengthened by the no detection of local expansions.

Conclusions

The interpolation analyses and complete sequencing of present mtDNA sub-Saharan lineages observed in North Africa support the genetic impact of recent trans-Saharan migrations, namely the slave trade initiated by the Arab conquest of North Africa in the seventh century. Sub-Saharan people did not leave traces in the North African maternal gene pool for the time of its settlement, some 40,000 years ago.
Ensembl variation resources
Authors: Chen Yuan , Cunningham Fiona , Rios Daniel , McLaren William M , Smith James , Pritchard Bethan , Spudich Giulietta M , Brent Simon , Kulesha Eugene , Marin-Garcia Pablo , Smedley Damian , Birney Ewan , Flicek Paul
Published: May 2010

Background

The maturing field of genomics is rapidly increasing the number of sequenced genomes and producing more information from those previously sequenced. Much of this additional information is variation data derived from sampling multiple individuals of a given species with the goal of discovering new variants and characterising the population frequencies of the variants that are already known. These data have immense value for many studies, including those designed to understand evolution and connect genotype to phenotype. Maximising the utility of the data requires that it be stored in an accessible manner that facilitates the integration of variation data with other genome resources such as gene annotation and comparative genomics.

Description

The Ensembl project provides comprehensive and integrated variation resources for a wide variety of chordate genomes. This paper provides a detailed description of the sources of data and the methods for creating the Ensembl variation databases. It also explores the utility of the information by explaining the range of query options available, from using interactive web displays, to online data mining tools and connecting directly to the data servers programmatically. It gives a good overview of the variation resources and future plans for expanding the variation data within Ensembl.

Conclusions

Variation data is an important key to understanding the functional and phenotypic differences between individuals. The development of new sequencing and genotyping technologies is greatly increasing the amount of variation data known for almost all genomes. The Ensembl variation resources are integrated into the Ensembl genome browser and provide a comprehensive way to access this data in the context of a widely used genome bioinformatics system. All Ensembl data is freely available at http://www.ensembl.org and from the public MySQL database server at ensembldb.ensembl.org.
Consistent annotation of gene expression arrays
Authors: Ballester Benoît , Johnson Nathan , Proctor Glenn , Flicek Paul
Published: May 2010

Background

Gene expression arrays are valuable and widely used tools for biomedical research. Today's commercial arrays attempt to measure the expression level of all of the genes in the genome. Effectively translating the results from the microarray into a biological interpretation requires an accurate mapping between the probesets on the array and the genes that they are targeting. Although major array manufacturers provide annotations of their gene expression arrays, the methods used by various manufacturers are different and the annotations are difficult to keep up to date in the rapidly changing world of biological sequence databases.

Results

We have created a consistent microarray annotation protocol applicable to all of the major array manufacturers. We constantly keep our annotations updated with the latest Ensembl Gene predictions, and thus cross-referenced with a large number of external biomedical sequence database identifiers. We show that these annotations are accurate and address in detail reasons for the minority of probesets that cannot be annotated. Annotations are publicly accessible through the Ensembl Genome Browser and programmatically through the Ensembl Application Programming Interface. They are also seamlessly integrated into the BioMart data-mining tool and the biomaRt package of BioConductor.

Conclusions

Consistent, accurate and updated gene expression array annotations remain critical for biological research. Our annotations facilitate accurate biological interpretation of gene expression profiles.
Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease
Authors: Sánchez-Mejías Avencia , Núñez-Torres Rocio , Fernández Raquel M , Antiñolo Guillermo , Borrego Salud
Published: May 2010

Background

Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach.

Methods

CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes.

Results

A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR.

Conclusions

The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.
Oleanane triterpenoid CDDO-Me induces apoptosis in multidrug resistant osteosarcoma cells through inhibition of Stat3 pathway
Authors: Ryu Keinosuke , Susa Michiro , Choy Edwin , Yang Cao , Hornicek Francis J , Mankin Henry J , Duan Zhenfeng
Published: May 2010

Background

The activation of signal transducer and activator of transcription 3 (Stat3) pathway correlates with tumor growth, survival, drug resistance and poor prognosis in osteosarcoma. To explore the potential therapeutic values of this pathway, we assessed both the expression and the activation of Stat3 pathway in several pairs of multidrug resistant (MDR) osteosarcoma cell lines, and tissues. To explore the potential therapeutic values of this pathway, we analyzed the ability of the synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), to inhibit Stat3 expression and activation as well as its effects on doxorubicin sensitivity in osteosarcoma cells.

Methods

Expression of Stat3, phosphorylated Stat3 (pStat3) and Stat3 targeted proteins, including Bcl-XL, Survivin and MCL-1 were determined in drug sensitive and MDR osteosarcoma cell lines and tissues by Western blot analysis. The effect of CDDO-Me on osteosarcoma cell growth was evaluated by MTT and apoptosis by PARP cleavage assay and caspase-3/7 activity.

Results

Stat3 pathway was activated in osteosarcoma tissues and in MDR cell lines. CDDO-Me inhibited growth and induced apoptosis in osteosarcoma cell lines. Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3. The inhibition of Stat3 pathway correlated with the suppression of the anti-apoptotic Stat3 targeted genes Bcl-XL, survivin, and MCL-1. Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.

Conclusions

Stat3 pathway is overexpressed in MDR osteosarcoma cells. CDDO-Me significantly inhibited Stat3 phosphorylation, Stat3 nuclear translocation and induced apoptosis in osteosarcoma. This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance.
Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification
Authors: Zhang Shuang , Wang Zhi Hui , Chen Guo Qiang
Published: May 2010

Background

PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.

Results

Fusion protein PhaR-intein-target protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and β-galactosidase (lacZ), were successfully purified using the PhaR based protein purification method.

Conclusion

The successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.
A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli
Authors: Banerjee Sampali , Kumar Jitendra , Apte-Deshpande Anjali , Padmanabhan Sriram
Published: May 2010

Background

The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.

Results

We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts.

Conclusions

This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.
Replication independent DNA double-strand break retention may prevent genomic instability
Authors: Kongruttanachok Narisorn , Phuangphairoj Chutipa , Thongnak Araya , Ponyeam Wanpen , Rattanatanyong Prakasit , Pornthanakasem Wichai , Mutirangura Apiwat
Published: May 2010

Background

Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs), and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs) located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, γ-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ), mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM)-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability.

Results

We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the γ-H2AX. When cells were treated with tricostatin A (TSA) and the histones became hyperacetylated, the amount of γ-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51.

Conclusions

Methylated RIND-EDSBs are retained in non-acetylated heterochromatin because there is a prolonged time lag between RIND-EDSB production and repair. The rapid cellular responses to DSBs may be blocked by compact heterochromatin structure which then allows these breaks to be repaired by a more precise ATM-dependent pathway. In contrast, Ku-mediated NHEJ can repair euchromatin-associated EDSBs. Consequently, spontaneous mutations in hypomethylated genome are produced at faster rates because unmethylated EDSBs are unable to avoid the more error-prone NHEJ mechanisms.
Hospital costs of central line-associated bloodstream infections and cost-effectiveness of closed vs. open infusion containers. The case of Intensive Care Units in Italy
Authors: Tarricone Rosanna , Torbica Aleksandra , Franzetti Fabio , Rosenthal Victor D
Published: May 2010

Objectives

The aim was to evaluate direct health care costs of central line-associated bloodstream infections (CLABSI) and to calculate the cost-effectiveness ratio of closed fully collapsible plastic intravenous infusion containers vs. open (glass) infusion containers.

Methods

A two-year, prospective case-control study was undertaken in four intensive care units in an Italian teaching hospital. Patients with CLABSI (cases) and patients without CLABSI (controls) were matched for admission departments, gender, age, and average severity of illness score. Costs were estimated according to micro-costing approach. In the cost effectiveness analysis, the cost component was assessed as the difference between production costs while effectiveness was measured by CLABSI rate (number of CLABSI per 1000 central line days) associated with the two infusion containers.

Results

A total of 43 cases of CLABSI were compared with 97 matched controls. The mean age of cases and controls was 62.1 and 66.6 years, respectively (p = 0.143); 56% of the cases and 57% of the controls were females (p = 0.922). The mean length of stay of cases and controls was 17.41 and 8.55 days, respectively (p < 0.001). Overall, the mean total costs of patients with and without CLABSI were € 18,241 and € 9,087, respectively (p < 0.001). On average, the extra cost for drugs was € 843 (p < 0.001), for supplies € 133 (p = 0.116), for lab tests € 171 (p < 0.001), and for specialist visits € 15 (p = 0.019). The mean extra cost for hospital stay (overhead) was € 7,180 (p < 0.001). The closed infusion container was a dominant strategy. It resulted in lower CLABSI rates (3.5 vs. 8.2 CLABSIs per 1000 central line days for closed vs. open infusion container) without any significant difference in total production costs. The higher acquisition cost of the closed infusion container was offset by savings incurred in other phases of production, especially waste management.

Conclusions

CLABSI results in considerable and significant increase in utilization of hospital resources. Use of innovative technologies such as closed infusion containers can significantly reduce the incidence of healthcare acquired infection without posing additional burden on hospital budgets.
Cancer stem cells in solid tumors: elusive or illusive?
Authors: Welte Yvonne , Adjaye James , Lehrach Hans R , Regenbrecht Christian RA
Published: May 2010

During the past years in vivo transplantation experiments and in vitro colony-forming assays indicated that tumors arise only from rare cells. These cells were shown to bear self-renewal capacities and the ability to recapitulate all cell types within an individual tumor. Due to their phenotypic resemblance to normal stem cells, the term "cancer stem cells" is used. However, some pieces of the puzzle are missing: (a) a stringent definition of cancer stem cells in solid tumors (b) specific markers that only target cells that meet the criteria for a cancer stem cell in a certain type of tumor. These missing parts started an ongoing debate about which is the best method to identify and characterize cancer stem cells, or even if their mere existence is just an artifact caused by the experimental procedures. Recent findings query the cancer stem cell hypothesis for solid tumors itself since it was shown in xenograft transplantation experiments that under appropriate conditions tumor-initiating cells are not rare.

In this review we critically discuss the challenges and prospects of the currently used major methods to identify cancer stem cells. Further on, we reflect the present discussion about the existence of cancer stem cells in solid tumors as well as the amount and characteristics of tumor-initiating cells and finally provide new perspectives like the correlation of cancer stem cells and induced pluripotent cells.
Experimental depletion of CD8+ cells in acutely SIVagm-Infected African Green Monkeys results in increased viral replication
Authors: Gaufin Thaidra , Ribeiro Ruy M , Gautam Rajeev , Dufour Jason , Mandell Daniel , Apetrei Cristian , Pandrea Ivona
Published: May 2010

Background

In vivoCD8+cell depletions in pathogenic SIV infections identified a key role for cellular immunity in controlling viral load (VL) and disease progression. However, similar studies gave discordant results in chronically-infected SMs, leading some authors to propose that in natural hosts, SIV replication is independent of cellular immunity. To assess the role of cellular immune responses in the control of SIV replication in natural hosts, we investigated the impact of CD8+cell depletion during acute SIV infection in AGMs.

Results

Nine AGMs were infected with SIVagm.sab and were followed up to day 225 p.i. Four were intravenously infused with the cM-T807 antibody on days 0 (50 mg/kg), 6, and 13 (10 mg/kg, respectively) post infection (p.i.). CD8+cells were depleted for up to 28 days p.i. in peripheral blood and LNs in all treated AGMs. Partial CD8+T cell depletion occurred in the intestine. SIVagm VLs peaked at similar levels in both groups (107-108RNA copies/ml). However, while VLs were controlled in undepleted AGMs, reaching set-point levels (104-105RNA copies/ml) by day 28 p.i., high VLs (>106RNA copies/ml) were maintained by day 21 p.i. in CD8-depleted AGMs. By day 42 p.i., VLs were comparable between the two groups. The levels of immune activation and proliferation remained elevated up to day 72 p.i. in CD8-depleted AGMs and returned to preinfection levels in controls by day 28 p.i. None of the CD8-depleted animals progressed to AIDS.

Conclusion

CD8+cells are responsible for a partial control of postacute viral replication in SIVagm.sab-infected AGMs. In contrast to macaques, the SIVagm-infected AGMs are able to control viral replication after recovery of the CD8+T cells and avoid disease progression.
The development of a rapid SYBR one step real-time RT-PCR for detection of porcine reproductive and respiratory syndrome virus
Authors: Tian Hong , Wu JingYan , Shang YouJun , Cheng Yan , Liu XiangTao
Published: May 2010

Background

Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak. In this study, a rapid SYBR-based, one step real-time RT-PCR quantitative reverse transcription PCR (qRT-PCR) has been developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Primers were designed based on the sequence of highly conservative region of PRRSV N gene.

Results

The sensitivity of the real-time qRT-PCR assay was achieved through PRRSV ch-1a RNA for the generation of a standard curve. The detection limit of the assay was found to be 9.6 RNA copies per reaction mixture. This assay had excellent intra- and inter-assay reproducibility as in total 65 field samples were screened for the presence of PRRSV by conventional RT-PCR in parallel with qRT-PCR, and the detection rate increased from 60.0% to 76.9%. Moreover, the specificity result indicated that this assay could reliably differentiate PRRSV from the other swine viral diseases, such as classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).

Conclusion

The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PRRSV in field samples.
Characterization of variants in the promoter of BZLF1 gene of EBV in nonmalignant EBV-associated diseases in Chinese children
Authors: Jin Yingkang , Xie Zhengde , Lu Gen , Yang Shuang , Shen Kunling
Published: May 2010

Background

Diseases associated with Epstein-Barr virus (EBV) infections, such as infectious mononucleosis (IM), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV) are not rare in Chinese children. The association of type 1 or type 2 EBV and variants of the EBV BZLF1 promoter zone (Zp) with these diseases is unclear.

Results

The objective of this study was to investigate the relationship between EBV genotypes (Zp variants and EBV type 1 and 2) and the clinical phenotypes of EBV-associated diseases in Chinese children. The Zp region was directly sequenced in 206 EBV-positive DNA samples from the blood of patients with IM, EBV-HLH, CAEBV, and healthy controls. Type 1 or type 2 EBV was examined by PCR for EBNA2 and EBNA3C subtypes. Four polymorphic Zp variants were identified: Zp-P, Zp-V3, Zp-P4 and Zp-V1, a new variant. The Zp-V3 variant was significantly associated with CAEBV (P ≤ 0.01). The frequency of co-infection with Zp variants was higher in patients with CAEBV and EBV-HLH, compared with IM and healthy controls, mostly as Zp-P+V3 co-infection. Type 1 EBV was predominant in all categories (81.3-95%) and there was no significant difference in the frequency of the EBV types 1 and 2 in different categories (P > 0.05).

Conclusions

Type 1 EBV and BZLF1 Zp-P of EBV were the predominant genotypes in nonmalignant EBV associated diseases in Chinese children and Zp-V3 variant may correlates with the developing of severe EBV infection diseases, such as CAEBV and EBV-HLH.
FoxP3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development
Authors: Haque Rizwanul , Lei Fengyang , Xiong Xiaofang , Wu Yuzhang , Song Jianxun
Published: May 2010

Introduction

Forkhead box p3 (FoxP3)-expressing regulatory T cells (Tregs) have been clearly implicated in the control of autoimmune disease in murine models. In addition, ectopic expression of FoxP3 conveys a Treg phenotype to CD4+ T cells, lending itself to therapeutic use in the prevention of rheumatoid arthritis (RA). In this study, we generated therapeutically active Tregs with an increased life span and hence greater therapeutic potential.

Methods

We used retrovirus-mediated transduction to introduce FoxP3 or FoxP3 with anti-apoptotic Bcl-2 family molecule Bcl-xL linked by a 2A picornavirus self-cleaving peptide into CD4+ T cells to generate Tregs. In addition, by using in vitro functional analyses and adoptive immunotherapy in a murine model of RA, we demonstrated that these Tregs were highly reactive.

Results

We found that CD4+ T cells expressing both FoxP3 and Bcl-xL were able to differentiate into functional Tregs, which have a long-term survival advantage over cells transduced with FoxP3 alone. In an in vivo murine model, adoptive transfer of Tregs expressing both FoxP3 and Bcl-xL demonstrated more effective suppression of RA than CD4+ T cells expressing FoxP3 alone.

Conclusions

FoxP3 and Bcl-xL can cooperatively promote the differentiation and persistence of Tregs, with the capacity to prevent arthritis. Our results provide a novel approach for generating highly reactive Tregs for augmenting cellular immunotherapy for autoimmune disease.