bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill
Authors: de Almeida Márcia R , Ruedell Carolina M , Ricachenevsky Felipe K , Sperotto Raul A , Pasquali Giancarlo , Fett-Neto Arthur G 
Published: October 2010

AbstractBackgroundEucalyptus globulusand its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting inE. globulusmicrocuttings.ResultsBy the use of two distinct algorithms,geNormandNormFinder, we have assessed gene expression stability of eleven candidate reference genes inE. globulus:18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBIand33380. The candidate reference genes were evaluated in microccuttings rootedin vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program:IDHandSANDforgeNorm, andH2BandTUAforNormFinder. Both programs indentifiedUBIand18Sas the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of theARGONAUTE1gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.ConclusionOur study showed that expression stability varied between putative reference genes tested inE. globulus. Based on theAGO1relative expression profile obtained using the genes suggested by the algorithms,H2BandTUAwere considered as the most suitable reference genes for expression studies inE. globulusadventitious rooting.UBIand18Swere unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.
A direct comparison of strategies for combinatorial RNA interference
Authors: Lambeth Luke S , Van Hateren Nick J , Wilson Stuart A , Nair Venugopal 
Published: October 2010

Abstract

Background
Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.

Results
Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.

Conclusions
By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.
Prevalence and novelty of PRPF31 mutations in French autosomal dominant rod-cone dystrophy patients and a review of published reports
Authors: Audo Isabelle , Bujakowska Kinga , Mohand-Saïd Saddek , Lancelot Marie-Elise , Moskova-Doumanova Veselina , Waseem Naushin H , Antonio Aline , Sahel José-Alain , Bhattacharya Shomi S , Zeitz Christina 
Published: October 2010

Abstract

Background
Rod-cone dystrophies are heterogeneous group of inherited retinal disorders both clinically and genetically characterized by photoreceptor degeneration. The mode of inheritance can be autosomal dominant, autosomal recessive or X-linked. The purpose of this study was to identify mutations in one of the genes, PRPF31, in French patients with autosomal dominant RP, to perform genotype-phenotype correlations of those patients, to determine the prevalence of PRPF31 mutations in this cohort and to review previously identified PRPF31 mutations from other cohorts.

Methods
Detailed phenotypic characterization was performed including precise family history, best corrected visual acuity using the ETDRS chart, slit lamp examination, kinetic and static perimetry, full field and multifocal ERG, fundus autofluorescence imaging and optic coherence tomography. For genetic diagnosis, genomic DNA of ninety families was isolated by standard methods. The coding exons and flanking intronic regions of PRPF31 were PCR amplified, purified and sequenced in the index patient.

Results
We showed for the first time that 6.7% cases of a French adRP cohort have a PRPF31 mutation. We identified in total six mutations, which were all novel and not detected in ethnically matched controls. The mutation spectrum from our cohort comprises frameshift and splice site mutations. Co-segregation analysis in available family members revealed that each index patient and all affected family members showed a heterozygous mutation. In five families incomplete penetrance was observed. Most patients showed classical signs of RP with relatively preserved central vision and visual field.

Conclusion
Our studies extended the mutation spectrum of PRPF31 and as previously reported in other populations, it is a major cause of adRP in France.
Expression profiling in canine osteosarcoma: identification of biomarkers and pathways associated with outcome
Authors: O'Donoghue Liza E , Ptitsyn Andrey A , Kamstock Debra A , Siebert Janet , Thomas Russell S , Duval Dawn L 
Published: October 2010

AbstractBackgroundOsteosarcoma (OSA) spontaneously arises in the appendicular skeleton of large breed dogs and shares many physiological and molecular biological characteristics with human OSA. The standard treatment for OSA in both species is amputation or limb-sparing surgery, followed by chemotherapy. Unfortunately, OSA is an aggressive cancer with a high metastatic rate. Characterization of OSA with regard to its metastatic potential and chemotherapeutic resistance will improve both prognostic capabilities and treatment modalities.MethodsWe analyzed archived primary OSA tissue from dogs treated with limb amputation followed by doxorubicin or platinum-based drug chemotherapy. Samples were selected from two groups: dogs with disease free intervals (DFI) of less than 100 days (n = 8) and greater than 300 days (n = 7). Gene expression was assessed with Affymetrix Canine 2.0 microarrays and analyzed with a two-tailed t-test. A subset of genes was confirmed using qRT-PCR and used in classification analysis to predict prognosis. Systems-based gene ontology analysis was conducted on genes selected using a standard J5 metric. The genes identified using this approach were converted to their human homologues and assigned to functional pathways using the GeneGo MetaCore platform.ResultsPotential biomarkers were identified using gene expression microarray analysis and 11 differentially expressed (p < 0.05) genes were validated with qRT-PCR (n = 10/group). Statistical classification models using the qRT-PCR profiles predicted patient outcomes with 100% accuracy in the training set and up to 90% accuracy upon stratified cross validation. Pathway analysis revealed alterations in pathways associated with oxidative phosphorylation, hedgehog and parathyroid hormone signaling, cAMP/Protein Kinase A (PKA) signaling, immune responses, cytoskeletal remodeling and focal adhesion.ConclusionsThis profiling study has identified potential new biomarkers to predict patient outcome in OSA and new pathways that may be targeted for therapeutic intervention.
PDGF-B-driven gliomagenesis can occur in the absence of the proteoglycan NG2
Authors: Terrile Marta , Appolloni Irene , Calzolari Filippo , Perris Roberto , Tutucci Evelina , Malatesta Paolo 
Published: October 2010

Abstract

Background
In the last years, the transmembrane proteoglycan NG2 has gained interest as a therapeutic target for the treatment of diverse tumor types, including gliomas, because increases of its expression correlate with dismal prognosis. NG2 has been shown to function as a co-receptor for PDGF ligands whose aberrant expression is common in gliomas. We have recently generated a glioma model based on the overexpression of PDGF-B in neural progenitors and here we investigated the possible relevance of NG2 during PDGF-driven gliomagenesis.

Methods
The survival curves of NG2-KO mice overexpressing PDGF-B were compared to controls by using a Log-rank test. The characteristics of tumors induced in NG2-KO were compared to those of tumors induced in wild type mice by immunostaining for different cell lineage markers and by transplantation assays in adult mice.

Results
We showed that the lack of NG2 does not appreciably affect any of the characterized steps of PDGF-driven brain tumorigenesis, such as oligodendrocyte progenitor cells (OPC) induction, the recruitment of bystander OPCs and the progression to full malignancy, which take place as in wild type animals.

Conclusions
Our analysis, using both NG2-KO mice and a miRNA based silencing approach, clearly demonstrates that NG2 is not required for PDGF-B to efficiently induce and maintain gliomas from neural progenitors. On the basis of the data obtained, we therefore suggest that the role of NG2 as a target molecule for glioma treatment should be carefully reconsidered.
Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide
Authors: Zhou Zhao-Li , Luo Zhi-Guo , Yu Bing , Jiang Yi , Chen Yi , Feng Jian-Ming , Dai Mei , Tong Lin-Jiang , Li Zheng , Li Yuan-Chao , Ding Jian , Miao Ze-Hong 
Published: October 2010

Abstract

Background
Hypoxia-inducible factor-1α (HIF-1α), a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α.

Results
Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide.

Conclusions
The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional activity, indicates the great therapeutic potential of triptolide as an anticancer drug. Meanwhile, our data further stress the possibility that HIF-1α functions in an unresolved nature or condition.
Improving the interpretation of quality of life evidence in meta-analyses: the application of minimal important difference units
Authors: Johnston Bradley C , Thorlund Kristian , Schünemann Holger J , Xie Feng , Murad Mohammad Hassan , Montori Victor M , Guyatt Gordon H 
Published: October 2010

Abstract
Systematic reviews of randomized trials that include measurements of health-related quality of life potentially provide critical information for patient and clinicians facing challenging health care decisions. When, as is most often the case, individual randomized trials use different measurement instruments for the same construct (such as physical or emotional function), authors typically report differences between intervention and control in standard deviation units (so-called "standardized mean difference" or "effect size"). This approach has statistical limitations (it is influenced by the heterogeneity of the population) and is non-intuitive for decision makers. We suggest an alternative approach: reporting results in minimal important difference units (the smallest difference patients experience as important). This approach provides a potential solution to both the statistical and interpretational problems of existing methods.
Estradiol, progesterone, testosterone profiles in human follicular fluid and cultured granulosa cells from luteinized pre-ovulatory follicles
Authors: Wen Xuesong , Li Dong , Tozer Amanda J , Docherty Suzanne M , Iles Ray K 
Published: October 2010

AbstractBackgroundThe production of sex steroids by follicular cells is proposed to be influenced by the maturity of the incumbent oocyte. Thus steroid levels may reflect suitability of an oocyte for IVF. We examined follicular fluids and granulosa cell production of steroid from IVF patients in order to test the relationship between steroid levels and fertilization.MethodsFollicular fluid and granulosa cells were extracted from 206 follicles of 35 women undergoing controlled ovarian stimulation. Follicular fluid was assayed for estradiol, progesterone and testosterone. Granulosa cells were cultured from individual follicles and their culture media assayed for production of these hormones after 24 hrs in vitro. Levels of steroids were correlated with follicular diameter, oocyte recovery and subsequent fertilization.ResultsFollicular fluid levels of progesterone were 6100 times higher than that of estradiol, and 16,900 times higher that of testosterone. Despite the size of follicle triggered after controlled luteinisation, the levels of progesterone and testosterone were maintained at relatively constant levels (median 98.1 micromoles/L for progesterone, and 5.8 nanomoles/L for testosterone). However, estradiol levels were slightly lower in the larger follicles (follicular diameter 10-15 mm, median 25.3 nanomoles/L; follicles > = 15 mm, median 15.1 nanomoles/L; linear correlation r = -0.47, p < 0.0001). With respect to oocyte recovery, no steroid showed a significant association in follicular fluid levels. Similarly no difference in follicular fluid steroid levels was found for those oocytes that did or did not fertilize. Significant quantities of progesterone were produced by the granulosa cells but production was constant regardless of the size of follicle from which the cells originated. Estradiol levels were only detectable in 10 of 121 cultures examined, and testosterone in none. Interestingly, when an oocyte was present follicular estradiol levels correlated with progesterone levels. However, when absent, follicular estradiol levels correlated with testosterone levels but not with progesterone.ConclusionsThe principle steroid product of luteinized pre-ovulatory granulosa is progesterone, a differentiation triggered by the gonadotropin surge. However, absolute steroid levels are associated with follicular size, not oocyte maturation/ability to fertilize.
Distinct modulation of microglial amyloid β phagocytosis and migration by neuropeptidesi
Authors: Fleisher-Berkovich Sigal , Filipovich-Rimon Talia , Ben-Shmuel Sarit , Hülsmann Claudia , Kummer Markus P , Heneka Michael T 
Published: October 2010

AbstractMicroglial activation plays an integral role in the development and course of neurodegeneration. Although neuropeptides such as bradykinin (BK), somatostatin (SST), and endothelin (ET) are known to be important mediators of inflammation in the periphery, evidence of a similar function in brain is scarce. Using immunocytochemistry, we demonstrate the expression of receptors for BK (B1, B2 subtypes), ET (ETA, ETB subtypes) and SST (SST 2, 3, 4 subtypes) in primary microglia and microglial cell lines. Exposure of BV2 and N9, as well as primary microglial cells to BK or SST increased Aβ uptake in a concentration-dependent manner, whereas endothelin decreased Aβ uptake. This was caused by increased phagocytosis of Aβ since the rate of intracellular Aβ degradation remained unaffected. All neuropeptides increased chemotactic activity of microglia. In addition, BK reduced Aβ-induced expression of proinflammatory genes including iNOS and COX-2. ET decreased the Aβ-induced expression of monocyte chemoattractant protein 1 and interleukin-6. These results suggest that neuropeptides play an important role in chemotaxis and Aβ clearance and modulate the brain's response to neuroinflammatory processes.
Cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging
Authors: Tripathy Debjani , Yin Xiangling , Sanchez Alma , Luo Jinhua , Martinez Joseph , Grammas Paula 
Published: October 2010

AbstractBackgroundMost neurodegenerative diseases are age-related disorders; however, how aging predisposes the brain to disease has not been adequately addressed. The objective of this study is to determine whether expression of proteins in the cerebromicrovasculature related to inflammation, oxidative stress and neurotoxicity is altered with aging.MethodsBrain microvessels are isolated from Fischer 344 rats at 6, 12, 18 and 24 months of age. Levels of interleukin (IL)-1β and IL-6 RNA are determined by RT-PCR and release of cytokines into the media by ELISA. Vessel conditioned media are also screened by ELISA for IL-1α, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α, (TNFα), and interferon γ (IFNγ). Immunofluorescent analysis of brain sections for IL-1β and IL-6 is performed.ResultsExpression of IL-1β and IL-6, both at RNA and protein levels, significantly (p < 0.01) decreases with age. Levels of MCP-1, TNFα, IL-1α, and IFNγ are significantly (p < 0.05-0.01) lower in 24 month old rats compared to 6 month old animals. Immunofluorescent analysis of brain vessels also shows a decline in IL-1β and IL-6 in aged rats. An increase in oxidative stress, assessed by increased carbonyl formation, as well as a decrease in the antioxidant protein manganese superoxide dismutase (MnSOD) is evident in vessels of aged animals. Finally, addition of microvessel conditioned media from aged rats to neuronal cultures evokes significant (p < 0.001) neurotoxicity.ConclusionsThese data demonstrate that cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging and suggest that the microvasculature may contribute to functional changes in the aging brain.
Factors that predict fertility desires for people living with HIV infection at a support and treatment centre in Kabale, Uganda
Authors: Kakaire Othman , Osinde Michael O , Kaye Dan K 
Published: October 2010

Abstract

Background
Studies from different contexts worldwide indicate that HIV positive patients manifest high-risk sexual behavior characterized by fertility intentions, multiple sexual partners, non-use of contraceptives and non-disclosure of HIV status to their sex partners. The objective was to analyze fertility desires among persons living with HIV at a treatment centre in Kabale Hospital, Southwestern Uganda.

Methods
From January to August 2009, we interviewed 400 HIV positive patients seeking care using an interviewer-administered questionnaire. We assessed socio-demographic variables, reproductive history, sexuality and fertility desires. At bivariate and multivariate analysis, characteristics of participants who reported or did not report desire to have a child in the near future were compared.

Results
Of the 400 respondents, (25.3%) were male, 47.3% were aged 25-34 years, over 85% were currently married or had ever been married, and the 62% had primary level of education or less. Over 17% had produced a child since the HIV diagnosis was made, and 28.6% reported that they would like to have a child in the near future. Age of the respondent, being single (versus being ever-married) and whether any of the respondents' children had died were inversely associated with fertility intentions.

Conclusion
Factors inversely associated with fertility intentions were age of the respondent, marital status and whether any of the respondents' children had died. Use of antiretroviral therapy was not associated with fertility intentions.