Search is not available for this dataset
text
stringlengths 1
134k
| id
stringlengths 11
16
| article_id
stringlengths 8
10
| section_title
stringlengths 1
1.35k
| educational_score
float64 0.52
5.16
| domain
stringclasses 3
values | document_type
stringclasses 4
values | domain_scores
listlengths 3
3
| document_type_scores
listlengths 4
4
| language
stringclasses 38
values | language_score
float64 0
1
|
|---|---|---|---|---|---|---|---|---|---|---|
To further characterize the mechanism (soluble factor versus membrane-bound factor) responsible for such an increase in astrocytes number in presence of npMSCs, we tried to induce the differentiation of neurospheres with npMSC conditioned medium. The following data were obtained: 77.5 ± 2.5% of the neurosphere-derived cells became GFAP-positive, 4.3 ± 0.9% were Tuj1-positive and 1.5 ± 0.6% were 04-positive . Conversely, in nnMSCs conditioned medium, differentiated phenotypes were distributed as follows: 56 ± 3.5%, 3.7 ± 1.3% and 5.9 ± 1.3% and were similar to results obtained in corresponding co-cultures. Moreover, we confirmed those results by absolute count: 1) in npMSCs conditioned medium 392/504 cells were GFAP-positive, 9/504 cells were O4-positive and 32/504 cells were Tuj1-positive ; 2) in nnMSCs conditioned medium 251/515 cells were GFAP-positive, 29/515 were O4-positive and 23/515 were Tuj1-positive; 3) in the control condition (DEM/F12 + B27), 211/531 cells were GFAP-positive, 27/531 cells were O4-positive and 167/531 cells were Tuj1-positive. Hence, regarding the phenotypic allocation, no significant difference is observed between neurosphere differentiated in co-culture and in conditioned medium. (Student T test, P > 0.05). This clearly suggest that npMSCs-derived soluble factor(s) is (are) responsible for an increase in the astrocytes number and a decrease in the neurons and oligodendrocytes numbers.
|
15369599_p5
|
15369599
|
MSCs effect on neurospheres-derived astrocytes number is due to a soluble factor
| 4.235378 |
biomedical
|
Study
|
[
0.9995203018188477,
0.00028122769435867667,
0.0001985526760108769
] |
[
0.9993094205856323,
0.00021534849656745791,
0.0004022821376565844,
0.0000728884624550119
] |
en
| 0.999996 |
As mentioned above, neurosphere derived-cells include neural stem cells and progenitors which are still proliferating but already committed to a given cell fate. We characterised the proportion of each progenitor cell-type present in our neurospheres. After dissociation, cells were allowed to adhere for one hour in coated dish and then fixed, labelled and counted. In those conditions we observed 14.4 ± 7.1% of GFAP-positive cells, 7.1± 4.1% of Tuj1-positive cells and 3.4 ± 1.6% of O4-positive cells .
|
15369599_p6
|
15369599
|
Nestin-positive MSC conditioned medium act on the GFAP-positve cell death
| 4.103381 |
biomedical
|
Study
|
[
0.9995555281639099,
0.00021769384329672903,
0.00022670626640319824
] |
[
0.9993433356285095,
0.0003331048646941781,
0.00026183208683505654,
0.0000617319019511342
] |
en
| 0.999997 |
In order to define if npMSCs conditioned medium has an instructive or/and a selective effect on neural cells, we analysed the proliferative capacity and the cell death in the differentiating neurospheres. The BrdU incorporation in differentiating neurosphere-derived cells cultivated in npMSCs- or in nnMSCs-conditioned medium or in control (non-conditioned) medium (DEM/F12 + B27) did not show any significant differences . These data rule out a proliferative effect on already committed astrocyte progenitors or an inhibition of cell proliferation in oligodendroglial and neuronal progenitors. Furthermore, using propidium iodide incorporations and counting, we quantified the cell death in GFAP-, Tuj1- and O4-positive cell population during the differentiation culture in the three conditioned media (by npMSCs, by nnMSCs and non-conditioned). After 48 hours, we observed a significant decrease of the number of GFAP-positive cells which have incorporated the propidium iodide in npMSCs-conditioned medium in comparison to the two other culture condition. This result suggests that npMSC conditioned medium partially inhibit the cell death in the GFAP-positive cell population. Nevertheless, no significant increase of cell death is observed in O4- and Tuj1-positive cell population (Student T test, p > 0.05). When propidum iodide incorporations are performed after four days of differentiation without renewing the conditioned media, no significant differences in the cell death could be observed in the three lineages, whatever the culture condition .
|
15369599_p7
|
15369599
|
Nestin-positive MSC conditioned medium act on the GFAP-positve cell death
| 4.169075 |
biomedical
|
Study
|
[
0.9994744658470154,
0.00030082400189712644,
0.00022475094010587782
] |
[
0.9993650317192078,
0.00015746630378998816,
0.0004121132369618863,
0.0000654865289106965
] |
en
| 0.999996 |
Previous reports have shown that several secreted growth factors, including BMP2, BMP4, LIF and CNTF stimulate the differentiation of cultured neural precursors into astrocytes . The expression of those cytokines was compared in nnMSCs and in npMSCs using quantitative RT-PCR. Compared to nnMSCs, we found that npMSCs slightly increased the expression level of BMP4 mRNA (190 ± 26%) while the BMP2 (36 ± 7%) and LIF (50 ± 10%) expression is slightly decreased . No significant difference was observed concerning the CNTF mRNA expression. Western blotting analyses of npMSCs, nnMSCs and neurosphere-derived cells conditioned media showed that only npMSCs release in their culture medium the mature and biologically-active form of BMP4 (27 kDa), although nnMSCs and neurosphere-derived cells expressed the biologically-inactive precursor form of BMP4 (57 kDa) . Finally, we observed that neurospheres-derived cells cultivated in npMSCs conditioned medium supplemented with an anti-BMP4 blocking antibodies, differentiate into 47.1 ± 1.5% GFAP-positive cells, 9.9 ± 0.9% Tuj1-positive cells and 5.7 ± 0.5% O4-positive cells . Statistical analyses did not show significant differences in GFAP- or O4-positive cells compared to control culture conditions (student T test, p > 0.05, n = 5). However, the number of Tuj1-positive cells remains significantly lower (***student T test, p < 0.001, n = 5). We thus conclude that the major glial effects (increase of astrocytes and decrease of oligodendrocytes) of the npMSCs on the neural progenitor differentiation is due to the release of biologically-active BMP4. The inhibition of neuronal differentiation should be attributed to another yet uncharacterized soluble factor(s).
|
15369599_p8
|
15369599
|
BMP4 is present in nestin-positive conditioned medium and is responsible for the increase of GFAP-positive cells in differentiating NSCs
| 4.448734 |
biomedical
|
Study
|
[
0.9994256496429443,
0.00038720533484593034,
0.000187137266038917
] |
[
0.998708963394165,
0.0003477031132206321,
0.0008076460217125714,
0.00013568365829996765
] |
en
| 0.999998 |
During the last few years, a number of studies have addressed the phenotypic plasticity of MSCs. Most of them were performed in vivo and demonstrated that environmental factors play important roles in determining the ability of grafted MSCs to adopt a neural-like phenotype. Grafting of a subset of MSCs into the lateral ventricle of neonatal mice resulted in their migration within the forebrain and cerebellum, and their differentiation into astrocytes . When MSCs were grafted into adult rat cerebellum after an ischemic lesion, they differentiated into cells expressing neuronal markers . Nakano et al. demonstrated that murine bone marrow cells differentiated into three distinct glial phenotypes (oligodendrocytes, astrocytes and microglia) when they were directly injected into the striatum of previously irradiated mice. Similarly, systemic injection of MSCs in lethally irradiated mice allowed their differentiation into neuronal and astroglial cells . Interestingly, the systemic injection of MSCs in non-irradiated but brain-lesioned mice had positive effects on injury repair, but very few MSC-derived cells expressed neural marker in such conditions . Given those results, somatic stem cells raise thus hopes in cell replacement strategies based on an autograft approach. Recent in vivo studies demonstrated that MSCs adopt neural fate by fusion with host neural cells. At least so far, there is no a clear and conclusive demonstration of a real differentiation of MSCs in neural cells. However, all those data obtained in vivo suggested that somatic stem cells seem to be promising for cellular therapy in neural diseases whatever the mechanism responsible for. It remains that a better knowledge of the molecular and cellular mechanism underlying the neural phenotypic plasticity of MSCs is required before considering those cells in human graft protocols.
|
15369599_p9
|
15369599
|
Discussion
| 4.4096 |
biomedical
|
Review
|
[
0.9982197880744934,
0.0009247853886336088,
0.000855528109241277
] |
[
0.31985926628112793,
0.0015017749974504113,
0.6779752373695374,
0.0006637352635152638
] |
en
| 0.999997 |
As we mentioned above, environment is able to modify the cellular differentiation capacity. In this study, we addressed the question of a possible effect of MSCs on neural progenitor cell fate and we choose an in vitro approach. Co-culture experiments demonstrate that MSCs display multiple activities in the regulation of embryonic striatum-derived progenitors and stem cells. Their effect mainly depends of their age in vitro : npMSCs (more than 10 passages in vitro or 25 doubling cell populations) increase the astrocytes numbers while inhibiting neuronal and oligodendroglial numbers. Conversely, nnMSCs (with a maximum of 5 passages in vitro ) only decrease the neuronal differentiation. Since similar results were obtained using MSC conditioned media, we concluded that these effects could be mediated by diffusible factor(s). We then analysed the effect of various conditioned media on the cell proliferation (using BrdU incorporation) as well as the cell death (using propidium iodide incorporation) in each neurosphere-derived cell types and at two different differentiation period (48 or 96 hours). We only observed a significant decrease of propidium iodide incorporation in GFAP-positive cell population at 48 hours of differentiation suggesting that soluble factor(s) select astroglial lineage by protecting those cells from cell death.
|
15369599_p10
|
15369599
|
Discussion
| 4.251172 |
biomedical
|
Study
|
[
0.9994390606880188,
0.00036625360371544957,
0.00019467588572297245
] |
[
0.999210000038147,
0.0002153601380996406,
0.000481932278489694,
0.0000927275832509622
] |
en
| 0.999996 |
The activity which drives neurosphere-derived cells into astrocytes only occurs in npMSC conditioned medium while the neuronal differentiation-inhibiting activity is present both in np- and nnMSC conditioned media. We tried to identify the astrogliogenetic factor(s) by measuring the expression by nn- and npMSCs of cytokines known to promote an astroglial fate . Our results show that only npMSCs express mature and biologically-active BMP4 while a biologically-inactive BMP4 precursor form is expressed and released by nnMSCs and neurosphere-derived cells in culture. Indeed, BMP4 is synthesized and released as an inactive precursor before being proteolytically activated by cleavage at the amino acid motif -Arg-Ser-Lys-Arg- . Relatively little is known about the regulatory mechanisms controlling the susceptibility of individual TGF-β family members to proteolytic cleavage. However, recent studies suggest that members of the subtilisin-like proprotein convertase (SPC) family, SPC1 and SPC4, could enhance the cleavage of BMP4 precursor. The availability of biologically active BMPs may therefore be controlled by the released of their precursor followed by the action of the proprotein convertases .
|
15369599_p11
|
15369599
|
Discussion
| 4.45334 |
biomedical
|
Study
|
[
0.9993853569030762,
0.00038175625377334654,
0.00023289602540899068
] |
[
0.9989653825759888,
0.0003885282785631716,
0.0005136111285537481,
0.00013252667849883437
] |
en
| 0.999998 |
The release by npMSCs of biologically-active form of BMP4 which promotes astroglial differentiation and inhibits oligodendroglial differentiation is consistent with previous studies demonstrating that BMPs play multiple roles in development . Other studies have reported that the effects of BMPs are age- and tissue-dependent and that BMPs promote astroglial differentiation and inhibit oligodendroglial differentiation when applied to cultures of cortical cells plated at E16 . Likewise, brief treatment with BMPs induces astroglial fate in cultured neural precursors from embryonic mice or in oligodendrocytes from newborn rats . More recently, Rajan et al. demonstrated that BMP4 induces astroglial differentiation of E14 and adult cortical neural stem cells from the subventricular zone when they are placed in high density culture. In this case, BMP4 has a true instructive role as NSCs cultures were clonal. Beside the instructive effect of BMP4 on glial lineages, some studies explained how BMP4 could act on astrocytic and oligodendrocytic precursors. A study realised on cerebellar primitive neurectodermal tumor cell line demonstrated that a high concentration of BMP2 and BMP4 attenuate apoptosis . BMP-mediated inhibition of oligodendrogenesis is controlled through the repression of the former transcription factor olig2 known to be essential for the oligodendrocytic development . All of these data suggest that BMP4 released by npMSCs selectively act on astrocytic and oligodendrocytic progenitors. In our experiments, we only demonstrate the anti-apoptotic effect of npMSCs-conditioned medium but we didn't test its possible instructive effect in clonal cultures. As BMP4, present in a biologically-active form in npMSCs conditioned medium, has been identified to be responsible of the increase of astrocytes numbers by the immunoblocking experiment and as it has been already demonstrated to be instructive , one could hypothesise that, in our system, the increase of astrocytes in response to npMSCs-derived BMP4 is a consequence of both a anti-apoptotic effect on GFAP-positive cells and also an instructive effect on NSCs.
|
15369599_p12
|
15369599
|
Discussion
| 4.58578 |
biomedical
|
Study
|
[
0.9992721676826477,
0.0004208858299534768,
0.00030697399051859975
] |
[
0.9983487129211426,
0.0005392484017647803,
0.0009442503214813769,
0.00016777012206148356
] |
en
| 0.999997 |
When considering the use of MSCs in cell replacement strategies for the treatment of various neurological diseases, it should be taken into account that those cells could also influence the development host neural precursors.
|
15369599_p13
|
15369599
|
Conclusions
| 3.263363 |
biomedical
|
Other
|
[
0.9983289837837219,
0.0005256357835605741,
0.0011453748447820544
] |
[
0.11924050003290176,
0.8679031133651733,
0.012051801197230816,
0.0008045839495025575
] |
en
| 0.999998 |
Adult rat bone marrow was obtained from femoral and tibial bones by aspiration and was resuspended into 5 ml of DEM (Invitrogen, Merelbeke, Belgium) . Between 100 and 200 × 10 6 marrow cells were plated on 175-cm 2 tissue culture flask in DEM/10% foetal bovine serum (Invitrogen). After 24 hours, the non-adherent cells were removed. When the rMSCs became confluent, they were resuspended with 0.25% Trypsin and 1 mM EDTA and then sub-cultured. Nestin expression by MSCs was induced as described in .
|
15369599_p14
|
15369599
|
Preparation and culture of rat mesenchymal stem cells (MSCs)
| 4.141823 |
biomedical
|
Study
|
[
0.9995531439781189,
0.00023528565361630172,
0.00021145066421013325
] |
[
0.9973862767219543,
0.002072797389701009,
0.00043379340786486864,
0.00010723601008066908
] |
en
| 0.999994 |
Green C57BL/6 mice embryos (Jackson Immunoresearch Laboratory, Inc., West Grove, USA) or NMRI mice were used as a source of striatal neural progenitor and stem cells. Green mouse express GFP under control of the β-actin promoter . The day of conception was determined by the presence of a vaginal plug (embryonic day 0). E16 striata were isolated and triturated in DEM/F12 (Invitrogen) with a sterile Pasteur pipette. The cell suspension was filtered with a 70 μm-mesh and viable cells were estimated by trypan blue exclusion. The cells were plated (1 × 10 6 cells/75-cm 2 tissue culture flask) in DEM/F12 supplemented with EGF (20 ng/ml, Sigma) and B27 (Invitrogen), a multi-component cell culture supplement devoid of any growth factor. When the size of neurospheres reached approximately 50 cells, they were dissociated into a single cell suspension by trituration and replated in fresh culture medium. Neurospheres with a maximum of 3 passages were used in this study.
|
15369599_p15
|
15369599
|
Preparation and culture of mouse striatal neural progenitor and stem cells
| 4.117763 |
biomedical
|
Study
|
[
0.9995988011360168,
0.00021400598052423447,
0.00018718074716161937
] |
[
0.9990785121917725,
0.000588590104598552,
0.0002646336506586522,
0.00006823740841355175
] |
en
| 0.999997 |
MSCs and neurospheres were plated on polyornithine coated dishes for 5 days, in DEM/F12 containing only B27 supplement and were then processed for immunocytochemical analysis as described below.
|
15369599_p16
|
15369599
|
Co-culture of MSCs and neurospheres
| 3.968323 |
biomedical
|
Study
|
[
0.9994736313819885,
0.00022706284653395414,
0.00029928196454420686
] |
[
0.9832932949066162,
0.01576365903019905,
0.0007286641048267484,
0.00021441896387841552
] |
en
| 0.999996 |
MSCs were placed in DEM/F12 medium supplemented with B27 (Invitrogen), during 3 days. The conditioned media were then filtered with 0.22 μm-pore filter before being replaced on plated neurospheres during 5 days.
|
15369599_p17
|
15369599
|
Culture of neurospheres in MSCs conditioned medium
| 4.003058 |
biomedical
|
Study
|
[
0.9993607401847839,
0.0002637817815411836,
0.00037556703318841755
] |
[
0.9454565048217773,
0.05304350703954697,
0.0011215154081583023,
0.00037847639760002494
] |
en
| 0.999997 |
The cultures were fixed with 4% (v/v) paraformaldehyde for 15 minutes at room temperature and washed 3 times in TBS buffer. They were then permeabilized in 1% Triton-X100 (v/v) for 15 minutes and washed 3 times in TBS buffer. Non-specific binding was blocked by a 1 hour treatment in TBST (TBS buffer with 0.1% Tween) containing defatted milk powder (30 mg/ml). The cells were then incubated for 1 hour at room temperature with either anti-glial fibrillary acidic protein (GFAP, Dako, mouse IgG, dilution 1:500), or Tuj1 , or O4 (Chemicon, mouse IgM, dilution 1:100) primary antibodies (diluted in blocking buffer). After 3 washes in TBS, cells were then incubated in FITC- or Cy5-conjugated anti-mouse IgG or IgM (Jackson Immunoresearch, 1:500) for 1 hour at room temperature and in the dark. The nuclei were stained with ethidium homodimer (0.2 μM, Sigma). The preparations were then mounted in Fluoprep™ (Biomerieux; France) and observed using a Bio-Rad MRC1024 laser scanning confocal microscope. The fraction of positive cells was determined by counting 10 non-overlapping microscopic fields for each coverslip in at least three separate experiments (n then corresponds to the number of coverslips).
|
15369599_p18
|
15369599
|
Immunocytochemistry
| 4.194911 |
biomedical
|
Study
|
[
0.9993662238121033,
0.0004374520212877542,
0.00019632975454442203
] |
[
0.9968705773353577,
0.0024344520643353462,
0.0005408655270002782,
0.00015418246039189398
] |
en
| 0.999996 |
After 24 hours or 3 days of culture, BrdU (20 μM, Sigma) which is a S-phase marker, or propidium iodide (400 mg/ml, Sigma) was added to the differentiating neurosphere cultures for 18 hours before fixation and staining. Tuj1, O4 and GFAP immuno-labellings were performed as described above. For BrdU labelling, coverslips were then post-fixed for 10 minutes in 4% (v:v) paraformaldehyde, incubated in HCl 1 N for 20 minutes at 37°C, washed with sodium perborate solution (50 mM, pH 8.5) and finally incubated with an anti-BrdU antibody for 1 hour at room temperature (Oxford, rat IgG, dilution 1:200) and Cy-5-conjugated anti-rat antibody, 1 hour at room temperature. The preparations were analysed as describe above.
|
15369599_p19
|
15369599
|
BrdU and propidium iodide incorporation
| 4.089776 |
biomedical
|
Study
|
[
0.9995394945144653,
0.00023814737505745143,
0.00022235258074942976
] |
[
0.9983421564102173,
0.0012227260740473866,
0.0003492364485282451,
0.00008588438504375517
] |
en
| 0.999997 |
Total RNA was prepared using the RNeasy total RNA purification kit (Qiagen, Westburg). For cDNA synthesis, random hexamer primers (Invitrogen) were used to prime reverse transcriptase reactions. The cDNA synthesis was carried out using Moloney-murine leukemia virus (M-MLV) Superscript II Reverse transcriptase (Invitrogen) following the manufacturer's instructions. Quantitative PCR was carried out using standard protocols with Quantitec SYBR Green PCR Kit (Qiagen). The PCR mix contained SYBR Greeen Mix, 0.5 μM primers (Table 1 ), 1 ng DNA template and nuclease free water to final volume of 25 μl. PCR were performed on RotorGene RG-3000 (Corbett Research) and analyzed with Rotorgene Software (Corbett Research). The percentage of gene expression by npMSCs was normalized in function of GAPDH gene expression and compared to the gene expression by nestin-negative MSCs that was considered as 100%. Each gene analysis was realised on three different samples with two run/sample (we then provided 6 PCR analyses by target gene)
|
15369599_p20
|
15369599
|
RNA extraction and quantitative RT-PCR analysis
| 4.147961 |
biomedical
|
Study
|
[
0.9995806813240051,
0.0002362211380386725,
0.00018314715998712927
] |
[
0.9990028738975525,
0.0006094877608120441,
0.0003132331767119467,
0.00007448594988090917
] |
en
| 0.999997 |
After 72 hours of culture, the conditioned medium (1.5 ml) from cultures of neural progenitor cells, npMSCs and nnMSCs were incubated with heparin sepharose CL-6B (Amersham Biosciences, Belgium) at 4°C for 24 hours. After centrifugation (700 × g), the supernatant was removed and bound proteins were eluted in loading buffer (glycerol 10% v/v; Tris 0.05 M pH 6.8; SDS 2%, bromophenol blue and 2.5% v/v – mercaptoethanol) by heating at 70°C for 10 minutes. The protein concentrations of various samples were quantified using the "RC DC Protein Assay" (Bio-Rad, Belgium) and equal protein quantities were loaded in each lane in a 15% sodium dodecyl sulfate polyacrylamide gel and electrophoresed. Then the proteins were transferred to PVDF membrane (Amersham Biosciences, Belgium) using a Trans-Blot Semi-Dry Transfer apparatus (Bio-Rad). The membranes were saturated with 3% gelatin (BioRad) during 1 hour at 37°C, then incubated for 1 hour with a monoclonal antibody against BMP4 (R&D, goat IgG, 0.1 μg/ml) or actin, as control for protein loading (Sigma, mouse IgG 1:5 000) at room temperature and then washed several times with PBS-0.1% Tween. The membrane was then incubated in Cy5-conjugated anti-goat IgG or anti-mouse IgG for 1 hour at room temperature, in the dark. After several washes in PBS, the membranes were scanned using a Typhoon 9200 Scanner (Amersham Biosciences) and subsequent analyses were performed with ImageQuant Software (Amersham Biosciences).
|
15369599_p21
|
15369599
|
Western Blot
| 4.170126 |
biomedical
|
Study
|
[
0.9994753003120422,
0.0003124898939859122,
0.00021219391783233732
] |
[
0.9984442591667175,
0.001031435327604413,
0.000432878325227648,
0.00009144430805463344
] |
en
| 0.999994 |
Neurospheres were plated on polyornithine-coated dishes and incubated during 5 days in DEM/F12 medium supplemented with B27 (Invitrogen) and previously conditioned by npMSCs during 3 days or not. Anti-BMP4 antibody (R&D, 2 μg/ml) was added on day 1 of this incubation.
|
15369599_p22
|
15369599
|
BMP4 neutralization
| 4.066046 |
biomedical
|
Study
|
[
0.9994866847991943,
0.00023218269052449614,
0.0002810989390127361
] |
[
0.9927142262458801,
0.0066687981598079205,
0.0004708334745373577,
0.00014605165051762015
] |
en
| 0.999996 |
SW performed most of the work and wrote a first draft of the manuscript; FB performed quantitative RT-PCR; GH, GM and PL were involved in the writing of the manuscript; BR conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.
|
15369599_p23
|
15369599
|
Authors' contributions
| 0.75139 |
other
|
Other
|
[
0.2976599335670471,
0.00447348365560174,
0.6978665590286255
] |
[
0.0054991659708321095,
0.9921342134475708,
0.0015815116930752993,
0.0007850737310945988
] |
en
| 0.999997 |
Postoperative administration of paracetamol or its prodrug propacetamol has been shown to decrease pain with a morphine sparing effect . The effect of propacetamol administered intraoperatively on postoperative pain and early postoperative morphine consumption has not been clearly evaluated. However, for a predictable moderate postoperative pain, intraoperative administration of non-opioid analgesics such as paracetamol and postoperative intravenous administration of morphine are recommended in patients undergoing general anesthesia with remifentanil . Indeed, remifentanil differs from potent mu agonists by its extremely short elimination half-life . The elimination kinetics of remifentanil is so fast that its analgesic effect wears off abruptly, thus making the management of postoperative pain critical.
|
15367329_p0
|
15367329
|
Background
| 3.987934 |
biomedical
|
Study
|
[
0.9966442584991455,
0.0029899768996983767,
0.00036586335045285523
] |
[
0.5079019665718079,
0.09215410053730011,
0.3974742293357849,
0.0024696714244782925
] |
en
| 0.999994 |
In order to evaluate the effectiveness of intraoperative paracetamol administration in the management of postoperative pain and morphine consumption, a standardized anesthesia protocol without long-acting opioid is necessary. Thus, for ethical reasons, the surgical procedure under general anesthesia with remifentanil as the only intraoperative analgesic must be associated with a moderate predictable postoperative pain.
|
15367329_p1
|
15367329
|
Background
| 3.925318 |
biomedical
|
Other
|
[
0.9890251755714417,
0.010130229406058788,
0.0008445985149592161
] |
[
0.3301878273487091,
0.6634188294410706,
0.0042519336566329,
0.002141432836651802
] |
en
| 0.999996 |
Therefore, the present study was designed to evaluate the effect of intraoperative administration of propacetamol during remifentanil-based anesthesia on postoperative pain in patients undergoing reduction mammoplasty.
|
15367329_p2
|
15367329
|
Background
| 4.041057 |
biomedical
|
Study
|
[
0.9872447848320007,
0.012195136398077011,
0.0005600946606136858
] |
[
0.990970253944397,
0.007639745716005564,
0.0005642626201733947,
0.0008256876026280224
] |
en
| 0.999997 |
After approval by the Local Ethical Committee and written informed consent, 36 consecutive female patients who underwent elective reduction mammoplasty were included. Exclusion criteria were the preoperative use of analgesic drugs; a body mass index ≥ 35, an American Society of Anesthesiology physical status ≥ 3 and sensitivity to paracetamol. Pain evaluation using a Simplified Numerical Pain Scale (SNPS) was explained to the patients during the preoperative anesthetic visit and the day before surgery. A standardized surgical technique was used for all patients.
|
15367329_p3
|
15367329
|
Patients
| 3.835487 |
biomedical
|
Study
|
[
0.7786692976951599,
0.22007311880588531,
0.00125756801571697
] |
[
0.9443923234939575,
0.03745564818382263,
0.0016312195220962167,
0.01652093604207039
] |
en
| 0.999997 |
Hydroxyzine 2 mg.kg -1 was given orally 12 h and 2 h before anesthesia as premedication. Before induction of anesthesia, patients were randomly allocated to either Placebo group or Propacetamol group. Randomization was based on computer-generated codes maintained in sequentially numbered, opaque envelopes. The preparation of the propacetamol or saline infusion was done by a nurse who was not in charge of the patient. The anesthetist, the patient and the nurse's staff caring for the patients in the recovery room were unaware of the treatment group. In both groups, anesthesia was induced with propofol 2.5 mg.kg -1 followed by a slow bolus (1 min) of remifentanil 1 mcg.kg -1 . Tracheal intubation was facilitated by atracurium 0.5 mg.kg -1 . Anesthesia was maintained with remifentanil 0.1 mcg.kg -1 .min -1 and isoflurane (0.5–1.0% end-tidal) with nitrous oxide (N 2 O) in 50% oxygen. Remifentanil infusion rate was increased or decreased by 0.05 mcg.kg.min -1 in order to maintain an arterial systolic pressure of 20% more or less than the baseline value. One hour before the end of surgery, which corresponded to the beginning of the skin closure, patients received either propacetamol 2 g in 50 cc saline (Propacetamol group) or 50 cc saline alone (Placebo group) infused over 10 min. At the end of surgery, administration of isoflurane and N 2 O were withdrawn and the patient was transferred in the recovery room. The anaesthetist, the patient and the nurse's staff caring for the patients in the recovery room were unaware of the treatment group. Remifentanil infusion was interrupted when the patient arrived in recovery room. Tracheal extubation was performed within a few minutes after remifentanil discontinuation. Clinical monitoring included heart rate, blood pressure, pulse oxymetry, respiratory rate, and sedation score (0: awake, 1: drowsy and 2: asleep).
|
15367329_p4
|
15367329
|
Anesthetic protocol
| 4.016133 |
clinical
|
Other
|
[
0.4075579345226288,
0.5881736874580383,
0.004268408287316561
] |
[
0.17901401221752167,
0.7682755589485168,
0.006069363094866276,
0.0466410368680954
] |
en
| 0.999997 |
From the time of extubation, pain was evaluated on using a SNPS (from 0, no pain to 10 the worse pain) and intravenous 2 mg morphine was administered on request every 5 min until pain relief (SNPS<4). When pain relief was reached, SNPS was subsequently evaluated every 15 min. Morphine was interrupted when the sedation score went up to 1, systemic arterial pressure < 80 mmHg or respiratory rate less than 8/min. During the data collection period, intravenous morphine titration was further administered if SNPS was up to 4. Patients did not receive antiemetic prophylaxis. If post-operative nausea and vomiting (PONV) occurred, metoclopramide 10 mg and ondansetron 4 mg if necessary were intravenously administered.
|
15367329_p5
|
15367329
|
Anesthetic protocol
| 3.918334 |
clinical
|
Study
|
[
0.390512079000473,
0.6069587469100952,
0.0025291561614722013
] |
[
0.5394922494888306,
0.39245548844337463,
0.005888903979212046,
0.0621633306145668
] |
en
| 0.999999 |
Patients fulfilling Aldrete criteria were discharged from recovery room.
|
15367329_p6
|
15367329
|
Anesthetic protocol
| 1.704002 |
clinical
|
Other
|
[
0.08324448019266129,
0.9109200239181519,
0.0058354889042675495
] |
[
0.09715642780065536,
0.6164204478263855,
0.007070713210850954,
0.2793523669242859
] |
en
| 0.999995 |
Morphine requirement, pain and sedation scores were measured every 5 min until pain relief was obtained. When SNPS was below 4 during 15 min, parameters were subsequently recorded every 15 min. Morphine side effects (nausea, vomiting, urinary retention, shivering and itching) and need for supplemental medications (e.g., antiemetics) were also recorded.
|
15367329_p7
|
15367329
|
Measurements
| 3.516015 |
biomedical
|
Study
|
[
0.623411238193512,
0.37442246079444885,
0.0021662351209670305
] |
[
0.8186832070350647,
0.1588515043258667,
0.003446565940976143,
0.019018759950995445
] |
en
| 0.999995 |
The total dose of morphine in recovery room was the primary end point. Pain scores after extubation and one hour after tracheal extubation, delay for morphine requirement, delay for pain relief and incidence of morphine side effects were recorded.
|
15367329_p8
|
15367329
|
Measurements
| 3.100954 |
clinical
|
Study
|
[
0.41273030638694763,
0.5834741592407227,
0.00379553297534585
] |
[
0.7378747463226318,
0.23426410555839539,
0.0026271273382008076,
0.02523401938378811
] |
en
| 0.999996 |
Data are expressed as mean (± standard deviation) for quantitative variables normally distributed, or otherwise as median (25th – 75th percentiles) when data were not normally distributed, and as percentage for categorical variables. Data were analyzed using Statview 5.0 software (SAS Institute Inc, USA). For intergroup comparisons, categorical variables were compared using the chi-squared test and continuous variables were compared using the Student t test or Mann-Whitney U test, as appropriate. A p value less than 0.05 was considered as significant. Anticipating a standard deviation of 2.49 , it was calculated that 15 patients at least were necessary to show a difference between groups in morphine consumption of 4 mg (considered as a clinically relevant difference) with a 80% power and a 5% type 1 error.
|
15367329_p9
|
15367329
|
Statistical analysis
| 4.049356 |
biomedical
|
Study
|
[
0.9981504082679749,
0.0016530416905879974,
0.00019658353994600475
] |
[
0.9937892556190491,
0.005488215014338493,
0.00044977356446906924,
0.0002726983220782131
] |
en
| 0.999996 |
Thirty-six patients were included during a 12 months period: 19 in Propacetamol group and 17 in Placebo group (Table 1 ). There was no significant difference between the groups concerning clinical characteristics, anesthesia duration and total amount of remifentanil administered. In all patients, extubation was obtained within 7 ± 3 min (Table 1 ) after remifentanil discontinuation.
|
15367329_p10
|
15367329
|
Results
| 3.907397 |
biomedical
|
Study
|
[
0.7610803246498108,
0.23703867197036743,
0.001881055417470634
] |
[
0.8910725712776184,
0.0888596922159195,
0.002093152143061161,
0.017974629998207092
] |
en
| 0.999996 |
In recovery room, cumulative morphine consumption was significantly lower in the Propacetamol group than in the Placebo group (Table 2 ). Five minutes after extubation, pain scores were similar in both groups (SNPS = 6). Pain scores one hour after tracheal extubation were significantly lower in the Propacetamol group than in the Placebo group (Table 2 ). Moreover, the time to reach a SNPS score less than 4 was significantly shorter in Propacetamol group compared to the Placebo group. All the patients received intravenous morphine titration in the first hour after extubation and three patients (one in Placebo group and two in Propacetamol group) required a morphine titration over one hour after extubation. Once a SNPS value less than 4 was obtained, pain scores remained stable and similar in both groups except in one patient in Placebo group who required an additional 2 mg intravenous bolus of morphine 80 min after extubation.
|
15367329_p11
|
15367329
|
Results
| 4.071452 |
biomedical
|
Study
|
[
0.8262766599655151,
0.17217940092086792,
0.001543897669762373
] |
[
0.9190313220024109,
0.06884356588125229,
0.0028728190809488297,
0.009252354502677917
] |
en
| 0.999998 |
The incidence of morphine adverse effects was similar in both groups: 5 patients had nausea (3 in Propacetamol and 2 in Placebo group, p=NS) and 4 patients had vomiting (2 in each group, p=NS), no other side effects were observed. In one patient receiving placebo, morphine titration was interrupted because of nausea and vomiting.
|
15367329_p12
|
15367329
|
Results
| 3.834517 |
biomedical
|
Study
|
[
0.6209094524383545,
0.3760876953601837,
0.0030028210021555424
] |
[
0.8164786696434021,
0.15928710997104645,
0.003535027150064707,
0.020699189975857735
] |
en
| 0.999996 |
This study shows that propacetamol administered one hour before end of surgery reduced the morphine dose given over the first four postoperative hours and shortened the elapsed time to obtain a SNPS under 4 in patients undergoing elective reduction mammoplasty. In our study, surgical technique and anesthetic protocol were similar in both groups. Remifentanil was the only analgesic used during anesthesia period and was interrupted before morphine administration. Thus, the beneficial effect on postoperative pain observed is clearly linked to intraoperative administration of propacetamol.
|
15367329_p13
|
15367329
|
Discussion
| 4.089947 |
biomedical
|
Study
|
[
0.9887477159500122,
0.01088351383805275,
0.00036864777212031186
] |
[
0.9946814179420471,
0.0036354241892695427,
0.0009159681503660977,
0.0007672054343856871
] |
en
| 0.999997 |
In a recent study , Verchère and colleagues failed to demonstrate a postoperative analgesic effect of intraoperative propacetamol administration, after remifentanil anesthesia for supratentorial craniotomy. Pain after supratentorial neurosurgery was too severe and paracetamol was insufficient to relief it. In our study mammoplasty was chosen because postoperative pain is moderate , and intravenous administration of morphine was used in recovery room. Thus, the morphine sparing effect of intraoperative administration of paracetamol could be really evaluated with respect of ethical requirement.
|
15367329_p14
|
15367329
|
Discussion
| 3.995124 |
biomedical
|
Study
|
[
0.9984038472175598,
0.0013479350600391626,
0.0002483004645910114
] |
[
0.9977565407752991,
0.0015455094398930669,
0.00048601077287457883,
0.00021192397980485111
] |
en
| 0.999997 |
Our results are apparently at variance with those of other previous studies. Paracetamol given rectally immediately after induction of anesthesia or at the end of gynecological surgery and orally before surgery failed to improve early postoperative analgesia. The negative results of these studies may be ascribable to several causes such as the low initial pain score in the control group , and the difference in the route used for paracetamol administration . An other hypothesis to consider is the use of long-acting opioid such as fentanyl that might have contributed to the early post-operative analgesia .
|
15367329_p15
|
15367329
|
Discussion
| 4.023539 |
biomedical
|
Study
|
[
0.9988775849342346,
0.0008949299808591604,
0.00022746609465684742
] |
[
0.9955565333366394,
0.0009058063733391464,
0.0033548225183039904,
0.00018279852520208806
] |
en
| 0.999997 |
Cobby and colleagues observed a morphine sparing effect when paracetamol 1.3 g was administered rectally at the end of hysterectomy . One explanation is that plasma concentration after 1.3 g paracetamol was sufficient to achieve opioid sparing effect comparable with that of intravenous propacetamol. This hypothesis is strengthened by the results of another trial showing that after rectal administration of a high dose of paracetamol (40 and 60.mg kg -1 ) pain score and analgesic demand were significantly reduced in the early postoperative period .
|
15367329_p16
|
15367329
|
Discussion
| 4.029245 |
biomedical
|
Study
|
[
0.9993001222610474,
0.0005019290838390589,
0.0001979484222829342
] |
[
0.9947468638420105,
0.0008365100366063416,
0.004286639392375946,
0.0001299236755585298
] |
en
| 0.999997 |
The presently observed incidence of postoperative nausea and vomiting was of 25% and was similar to that of a previous study . Despite the reduced dose of postoperative morphine in the Propacetamol group, the incidence of postoperative nausea and vomiting was not diminished. Our results are in agreement with the recent study of Aubrun and co-workers who observed that postoperative intravenous propacetamol allowed a morphine-sparing effect but did not reduce the incidence of morphine-related adverse effects in patients undergoing general surgery.
|
15367329_p17
|
15367329
|
Discussion
| 4.115748 |
biomedical
|
Study
|
[
0.9948166012763977,
0.004882988054305315,
0.0003003761812578887
] |
[
0.9966953992843628,
0.002296023303642869,
0.0006766112637706101,
0.0003319701354485005
] |
en
| 0.999999 |
Propacetamol was administered at the beginning of skin closure, which corresponds to one hour before the end of surgery. This delay may be insufficient to achieve pain control immediately after tracheal extubation, as the peak effect of intravenous propacetamol was shown to occur only two hours after its administration . Nevertheless, we considered that it was not feasible to administer propacetamol earlier, as in our practice duration of surgery was unpredictable.
|
15367329_p18
|
15367329
|
Discussion
| 2.943173 |
biomedical
|
Study
|
[
0.7390122413635254,
0.25841304659843445,
0.00257474509999156
] |
[
0.4981197714805603,
0.44318604469299316,
0.002440797397866845,
0.05625348165631294
] |
en
| 0.999998 |
In summary, intraoperative propacetamol administration in women undergoing reduction mammoplasty improved significantly early postoperative pain in recovery room, and should be recommended for postoperative pain management.
|
15367329_p19
|
15367329
|
Discussion
| 3.964188 |
biomedical
|
Study
|
[
0.9842430353164673,
0.015273998491466045,
0.00048292160499840975
] |
[
0.8023268580436707,
0.1665782630443573,
0.02605941891670227,
0.005035491660237312
] |
en
| 0.999997 |
The pre-publication history for this paper can be accessed here:
|
15367329_p20
|
15367329
|
Pre-publication history
| 1.031347 |
other
|
Other
|
[
0.013091341592371464,
0.0014214670518413186,
0.9854872226715088
] |
[
0.0015875872923061252,
0.997281551361084,
0.0006836647517047822,
0.0004471398133318871
] |
en
| 0.999997 |
Echocardiography allows the analysis of ventricular motion and heart-valve morphology. The discovery that injection of small air bubbles dispensed in saline are capable of causing an opacification of the heart started an enormous effort to develop contrast agents to assist in diagnosing coronary artery disease and myocardial infarction . Early developments led to stabilized bubbles in hyperosmolar solutions . Albumin-stabilized bubbles were one of the first contrast agents to allow examination of both ventricles , but there are stability problems in patients with elevated pulmonary pressure . Second-generation contrast agents improved the contrast especially in patients with subnormal echo signals . LK565 and comparable substances represent a new concept in contrast agents as it consists of small, stable, air-filled particles that provides a good contrast in both ventricles . Due to its high stability, LK565 improves examination of cardiac morphology and function as well as extracardiac vessel abnormalities for at least 15 minutes .
|
15357870_p0
|
15357870
|
Background
| 4.089086 |
biomedical
|
Review
|
[
0.9988840222358704,
0.0006253368919715285,
0.0004905425012111664
] |
[
0.18196256458759308,
0.009613999165594578,
0.8073539733886719,
0.0010694492375478148
] |
en
| 0.999997 |
The particles consist of a synthetic polymer of aspartic acid. Via peptide bonds, the molecules form bold, stable beads filled with air. The average size of LK565 particles is 3 μm in diameter . Because of their size and even polymeric surface the beads have similarities to microorganisms. This circumstance implies the induction of an immune response, which needs to be considered.
|
15357870_p1
|
15357870
|
Background
| 2.850681 |
biomedical
|
Other
|
[
0.9940658807754517,
0.0004768560756929219,
0.005457285325974226
] |
[
0.31378185749053955,
0.6833975315093994,
0.0018427122849971056,
0.0009778818348422647
] |
en
| 0.999998 |
Results from previous studies indicate that phagocytes are responsible for eliminating the contrast agent from the blood stream . In vitro, it has been shown that macrophages and neutrophils quickly devour the LK565 particles. Preliminary studies proved that the LK565 particles are phagocytosed by macrophages, resulting in slight activation with an increase in the intracellular calcium content. However, there were no symptoms of a respiratory burst .
|
15357870_p2
|
15357870
|
Background
| 4.008616 |
biomedical
|
Study
|
[
0.9995312690734863,
0.00020307974773459136,
0.00026571727357804775
] |
[
0.9990940093994141,
0.0005966007011011243,
0.00024038985429797322,
0.00006902038876432925
] |
en
| 0.999996 |
The activation of macrophages can lead to different conditions: no activation, normal activation, and overreaction. In the last case, over-expression of tumor necrosis factor alpha (TNF-α) can lead to a septic shock by induction of intravascular blood clots apart from other effects, which may result in disseminated intravascular coagulation . Furthermore, macrophages act as antigen-presenting cells which are highly important to induce an adaptive immune response, i.e. specific antibody production. It is probable that the LK565 particles attract antigen-presenting cells to present LK565 fragments as antigens. This may induce the production of specific antibodies against the contrast agent, which in rare cases could result in pathogenic immune reactions. The aim of this study was to examine a possible immune response to LK565 considering phagocytosis, TNF-α secretion by macrophages, lymphocyte activation and specific antibody production.
|
15357870_p3
|
15357870
|
Background
| 4.127325 |
biomedical
|
Study
|
[
0.9994335770606995,
0.00029050340526737273,
0.00027589770616032183
] |
[
0.9994809031486511,
0.00026755910948850214,
0.00019232489285059273,
0.00005927473466726951
] |
en
| 0.999996 |
The experiment was approved by the local ethics committee (registration number 708/98). After medical examination, 15 healthy male volunteers under the age of 30 were enrolled in this phase one clinical study. Each volunteer was exposed to a single dose of the contrast agent LK565 applied intravenously. Four participants were exposed to the contrast agent LK565 for the second time because they had already been involved in a prior study . The 15 volunteers were divided up into 3 groups (n = 5). The first group received 0.15 mg/kg bodyweight, the second group 0.4 mg/kg bodyweight and finally the third group 0.7 mg/kg LK565.
|
15357870_p4
|
15357870
|
Volunteers
| 3.745447 |
biomedical
|
Other
|
[
0.6284356713294983,
0.36784958839416504,
0.003714735619723797
] |
[
0.3758154809474945,
0.5989787578582764,
0.0024902469012886286,
0.0227154903113842
] |
en
| 0.999996 |
Prior to application, the contrast agent LK565 was dissolved in 10 ml physiological sodium chloride solution (37°C) under sterile conditions. To eliminate aggregations, the solution was filtered using a 20 μm mesh just before intravenous injection.
|
15357870_p5
|
15357870
|
Contrast agent
| 3.807431 |
biomedical
|
Study
|
[
0.9982434511184692,
0.0014730860712006688,
0.00028340218705125153
] |
[
0.8610872030258179,
0.13606509566307068,
0.001118847168982029,
0.0017288849921897054
] |
en
| 0.999998 |
Heparinized blood samples (2 ml) were taken before application (t = 0), after 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 1 ml after 10 d, 12 d and 14 d. For routine analysis, additional blood samples were drawn before and 2 hours after intravenous contrast agent injection. The volunteers were under medical care throughout the whole experiment.
|
15357870_p6
|
15357870
|
Sampling
| 3.559616 |
biomedical
|
Study
|
[
0.9971135854721069,
0.00252595916390419,
0.0003603429067879915
] |
[
0.995385468006134,
0.003898909315466881,
0.00034369967761449516,
0.0003719137457665056
] |
en
| 0.999997 |
Phagocytosis capacity was determined in vitro before (t = 0) and after 2 h, 6 h and 24 h with Phagotest™ (Becton Dickinson, Germany) and analyzed via flow cytometry (FACScalibur™, BectonDickinson, Germany). FITC-labeled E. coli, which were opsonized with antibodies and complement factors, were incubated with leukocytes. After incubation, the increased fluorescence of the phagocytes was due to the uptake of bacteria. The fluorescence intensity is dependent on the amount of phagocytosed bacteria. A sample chilled on ice was used for control.
|
15357870_p7
|
15357870
|
Phagocytosis
| 4.096959 |
biomedical
|
Study
|
[
0.9995102882385254,
0.0003064097836613655,
0.00018327892757952213
] |
[
0.9992208480834961,
0.0004005281371064484,
0.0003082095936406404,
0.00007048447878332809
] |
en
| 0.999998 |
Intracellular TNF-α production in monocytes and macrophages was analyzed before (t = 0) and after 2 h, 6 h, and 24 h. For detection, an anti-human TNF-α Pycoerythrin (PE) monoclonal antibody (mAb) was applied (Pharmingen, Germany). The specificity of the antibody had been validated in blocking experiments. To 40 μl blood, 145 μl RPMI and 15 μl 40 μM Monensin were added (Monensin prevents cytokine release by blocking the golgi apparatus). Cells were incubated for 2 h at T = 37°C and 5% CO 2 atmosphere. After washing, monocytes were surface stained by anti-human CD14 TriColor mAb (Medac, Germany). Red blood cells were lysed using FACS™ Lysing Solution (Becton Dickinson, Germany). Cells were fixed and permeabilized by addition of 250 μl Cytofix/Cytoperm™ (Becton Dickinson, Germany). Finally, the retained intracellular TNF-α was stained by the anti-TNF-α PE mAb. For a positive control, cells were stimulated with 0.25 μg Lipopolysaccharide (LPS), following the same procedure as mentioned above.
|
15357870_p8
|
15357870
|
Tumor necrosis factor alpha (TNF-α)
| 4.139207 |
biomedical
|
Study
|
[
0.9994789958000183,
0.00031587170087732375,
0.0002051846095127985
] |
[
0.9992238283157349,
0.00038670041249133646,
0.0003208191192243248,
0.00006863419548608363
] |
en
| 0.999997 |
Surface activation markers were analyzed before (t = 0) and after 2 h, 6 h, 24 h, 48 h, 72 h, and 96 h. For the detection of activation markers, anti-human CD69 PE (Holzel Diagnostika, Germany), CD25 Fluoresceinisothiocyanat (FITC), CD71 FITC, CD11b PE (Diaclone Research, Germany) and HLA-DR PerCP (Becton Dickinson, Germany) mAbs were used. For a positive control, cells were stimulated with 10 ng/ml Phorbol-12-Myristat-13-Acetat (PMA) and 1 μg/ml Ionomycin for 2 h, while macrophages/monocytes were stimulated with 1.25 μg/ml LPS for 2 h. To distinguish between leukocytes, additional surface markers, anti-human CD45 Allophycocyanine (APC), CD19 APC (Caltag Laboratories, Germany), CD3 Tri/Color (DAKO, Germany) and CD14 FITC (Diaclone Research, Germany) mAbs were used for detection. Blood samples (40 μl) were stained for 4-color flow cytometry analysis. After staining, red blood cells were lysed, subsequently leukocytes were analyzed. Antibody specificity was validated by isotype controls (Becton Dickinson, Germany).
|
15357870_p9
|
15357870
|
Surface activation markers
| 4.109976 |
biomedical
|
Study
|
[
0.99955815076828,
0.00023923102708067745,
0.00020261957251932472
] |
[
0.9992356300354004,
0.0003580382908694446,
0.0003479415609035641,
0.00005845662235515192
] |
en
| 0.999996 |
Serum samples were analyzed before (t = 0) and after 24 h, 48 h, 72 h, 96 h, 10 d, 12 d, and 14 d. Specific anti-LK565 antibodies were determined via indirect ELISA. Indirect ELISA was performed in a 96-well plate by LK565 immobilization overnight. Optimal concentrations had previously been determined by common cross-testing. Serum was obtained from blood samples after centrifugation at 400 G. After incubation for 2 h at room temperature, detector antibodies (Biozol Diagnostica GmbH, Germany) were added and incubated at 4°C overnight. Washing was carried out with phosphate buffered saline (PBS) and blocking buffer. For detection, alkaline phosphatase (AP)-linked goat-anti-human IgG and IgM antibodies with para-Nitrophenylphosphate (p-NPP) as substrate (Biozol Diagnostica GmbH, Germany) were used. After development, the enzyme reaction was stopped with 0.5 M NaOH and measured at λ = 405 nm. For the functional (positive) control of the indirect ELISA, total immunoglobuline was captured polyclonally. For a negative control, the serum of a person who had not been exposed to the contrast agent was obtained.
|
15357870_p10
|
15357870
|
Specific antibody development
| 4.123978 |
biomedical
|
Study
|
[
0.9993667006492615,
0.00039862547419033945,
0.00023469835286960006
] |
[
0.9993754029273987,
0.0002463152923155576,
0.00030704494565725327,
0.00007135473424568772
] |
en
| 0.999995 |
We investigated 15 volunteers in a clinical phase one trial over a period of 14 days. The kinetics of activation surface markers CD69, CD25, CD71 and CD11b on leukocytes, the phagocytosis capacity, and TNF-α production in monocytes were analyzed, as was the specific antibody production after LK565 application.
|
15357870_p11
|
15357870
|
Results
| 4.082557 |
biomedical
|
Study
|
[
0.9917033910751343,
0.007819940336048603,
0.0004767063073813915
] |
[
0.9856860041618347,
0.013113433495163918,
0.0004673001531045884,
0.0007332198438234627
] |
en
| 0.999998 |
The expression of CD69 , CD25 , CD71, HLA-DR , and CD11b is increased after activation . CD69 will be presented quickly after activation on almost all leukocytes normally after some hours but on neutrophils even after a few minutes due to mobilisation of intracellular storages . The α-chain of the IL-2 receptor CD25, also known as the high-affinity receptor on lymphocytes, is developed after 6 hours at the earliest and up to 1–2 days. Transferrin receptor CD71 correlates directly with cell proliferation and a late marker which can be expected after 3–4 days similar to antigen presentation via MHC-II analyzed as HLA-DR. Integrin expression (CD11b) on monocytes and neutrophils is associated with phagocyte migration into tissue .
|
15357870_p12
|
15357870
|
Results
| 4.364169 |
biomedical
|
Study
|
[
0.999431312084198,
0.00019260535191278905,
0.00037606863770633936
] |
[
0.9715831279754639,
0.02143079601228237,
0.006627485156059265,
0.00035868678241968155
] |
en
| 0.999997 |
Independent of dosage or repeated contact, the contrast agent LK565 was well tolerated by all volunteers. After exposure to LK565 no erythema, no drop in blood pressure, neither significant change of the heart frequency nor fever occurred (data not shown). The volunteers did not complain of relevant clinical symptoms such as chest discomfort or asthenia. It exhibited good opacification of both ventricles a few seconds after application . While the minimum dosage of 0.15 mg/kg LK565 was sufficient for just one echocardiogram, a suitable duration of echo contrast was obtained at a 0.4 mg/kg dose. A further increase in dosage provided no improvement in quality or contrast duration (data not shown).
|
15357870_p13
|
15357870
|
Results
| 4.123689 |
biomedical
|
Study
|
[
0.9905912280082703,
0.009103778749704361,
0.0003050531377084553
] |
[
0.9942723512649536,
0.0040920027531683445,
0.0007731106597930193,
0.0008625055779702961
] |
en
| 0.999997 |
The uptake of LK565 led to the saturation of phagocytes. The uptake capacity of the phagocytes from blood samples 6 h after injection was exhausted . Neither macrophages nor neutrophils phagocytosed any more labeled bacteria after LK565 uptake, the observed effect was reversible. After 24 h the phagocytic capacity reached almost the starting level.
|
15357870_p14
|
15357870
|
Results
| 3.887552 |
biomedical
|
Study
|
[
0.9993997812271118,
0.00027661866624839604,
0.0003236292104702443
] |
[
0.9978421926498413,
0.0016374108381569386,
0.0003961928014177829,
0.000124117752420716
] |
en
| 0.999999 |
During the first 24 h an increase in intracellular TNF-α production was detected in monocytes and macrophages . However, in relation to the positive control after 2 h of LPS stimulation, the slight increase in intracellular TNF-α was negligible. No connection between repeated exposure and dosage was found. The amount of intracellular TNF-α decreased to starting levels after 24 h.
|
15357870_p15
|
15357870
|
Results
| 4.018179 |
biomedical
|
Study
|
[
0.9994137287139893,
0.0002658107259776443,
0.00032052621827460825
] |
[
0.9991760849952698,
0.0004956143675372005,
0.0002617038553580642,
0.00006659965583821759
] |
en
| 0.999997 |
An increase in integrin expression CD11b was detected on monocytes and macrophages 6 hours after application. Compared to the positive control, the upregulation of the integrin was only poor. In all cases, the upregulation of the integrin was only of brief duration and returned to normal levels after 24 h . Only a slight and short increase in CD69 after 6 h was determined in neutrophils in a few cases. Most volunteers exhibited no CD69 increase in neutrophils. Even less CD69 expression was found on monocytes and macrophages where CD69 stimulation is associated with the production of prostaglandines, leukotriens and TNF-α. Only minor CD69 expression was observed on T-cells and on B-cells . No increase of CD25 and CD71 (also on monocytes) was detected .
|
15357870_p16
|
15357870
|
Results
| 4.174901 |
biomedical
|
Study
|
[
0.9994353652000427,
0.00033023461583070457,
0.00023434073955286294
] |
[
0.9994033575057983,
0.0002285584923811257,
0.000301075546303764,
0.000067011387727689
] |
en
| 0.999999 |
Discrete MHC class II (HLA-DR) upregulation on macrophages and lymphocytes was observed . We found no further evidence of an adaptive immune response via antibody production. None of the serum samples during our clinical trial exhibited any development of specific antibodies (IgM, IgG) versus the contrast agent .
|
15357870_p17
|
15357870
|
Results
| 3.618923 |
biomedical
|
Study
|
[
0.9876827597618103,
0.011533176526427269,
0.0007840850739739835
] |
[
0.9211307764053345,
0.07494251430034637,
0.0007456872845068574,
0.003181047970429063
] |
en
| 0.999996 |
The results show that there is only a slight activation of leukocytes esp. of phagocytes after application of LK565, a particular contrast agent based on a polymer of the naturally occurring amino aspartic acid. No specific antibody development against the contrast agent was detected. Thus, the risk of a major immunological activation cascade after repeated dosage can be regarded as minor. Since LK565 does not lead to an adaptive immune activation in this study, the possibility of an overexpression of IgE, which plays a key role in allergic diseases, also becomes more unlikely. Although none of the 15 volunteers enrolled in this phase I study developed an allergic-type reaction, such reactions could, however, occur in further phases of clinical testing or application. An allergic reaction against a pharmaceutical drug is a rare event which is impossible to exclude even in large clinical studies.
|
15357870_p18
|
15357870
|
Discussion
| 4.147456 |
biomedical
|
Study
|
[
0.9980179071426392,
0.0017800467321649194,
0.00020199851132929325
] |
[
0.9968717694282532,
0.002419494790956378,
0.0004218917165417224,
0.0002868695301003754
] |
en
| 0.999997 |
An increase in CD11b/CD18 is connected with phagocytosis and the diapedesis readiness of phagocytes. LK565 is eliminated from the blood stream via phagocytosis, therefore neutrophils exhibited an increase in CD11b expression and phagocytosis activity. This is also connected to a temporary higher intracellular TNF-α level in monocytes.
|
15357870_p19
|
15357870
|
Discussion
| 3.937052 |
biomedical
|
Study
|
[
0.9992974996566772,
0.00018736130732577294,
0.000515067542437464
] |
[
0.9818613529205322,
0.017462903633713722,
0.00042315840255469084,
0.00025266752345487475
] |
en
| 0.999996 |
On lymphocytes, especially T-cells, the cascade of CD69, CD25 and CD71 is closely related to an immune response. After expression of CD69, the interlinkage of the receptor leads to proliferation and secretion of IL-2, IFN-γ and TNF-α. After effective activation, the T-cells secrete IL-2 which activates the development of the high-affinity IL-2 receptor CD25 and therefore launches cell proliferation with an expression of CD71 after 3 – 4 days. During our study after LK565 application no major activation cascade was detected on either T-cells or B-cells.
|
15357870_p20
|
15357870
|
Discussion
| 4.170141 |
biomedical
|
Study
|
[
0.9995589852333069,
0.0001444731024093926,
0.00029656055266968906
] |
[
0.9975318908691406,
0.001960441702976823,
0.000426694838097319,
0.00008100400009425357
] |
en
| 0.999996 |
As mentioned above, the best contrast performance for echocardiography was obtained at a LK565 dosage of 0.4 mg/kg. Opacification of both ventricles was not enhanced by a further increase in dosage. Bearing in mind the fact that the contrast agent is phagocytosed and uptake capacity is limited, it is recommended that the daily dosage does not exceed 90 mg, which is equivalent up to three echocardiograms per day.
|
15357870_p21
|
15357870
|
Discussion
| 4.052361 |
biomedical
|
Study
|
[
0.9951878786087036,
0.004582745023071766,
0.00022928700491320342
] |
[
0.8903205990791321,
0.10040409862995148,
0.006840345449745655,
0.0024349018931388855
] |
en
| 0.999998 |
To minimize the risk of an undesirable adverse event such as an anaphylactoid reaction, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent (UCA) with a comfortable duration of signal enhancement esp. in echocardiography should be encouraged as a means to provide additional diagnostic information without causing a major activation cascade or triggering an adaptive immune response. This can be an advantage for the "difficult-to-image" patient with adverse reactions related to other UCAs.
|
15357870_p22
|
15357870
|
Conclusions
| 3.95551 |
biomedical
|
Other
|
[
0.9938231706619263,
0.005580472759902477,
0.0005964442971162498
] |
[
0.06955555081367493,
0.9139238595962524,
0.014500104822218418,
0.0020205441396683455
] |
en
| 0.999997 |
H.K. Maerz received salary for 6 months from Dr. F. Koehler Chemie GmbH, Alsbach-Haehnlein, Germany, the developer/manufacturer of LK565. R. Zotz is co-author of one of several patents related to LK565 (Zotz R, Erbel R, Krone V, Magerstädt M, Walch A : Ultrasonic contrast agents, processes for their preparation and the use thereof as diagnostic and therapeutic agents. United States Patent 5.137.928).
|
15357870_p23
|
15357870
|
Competing interests
| 1.073563 |
other
|
Other
|
[
0.029316749423742294,
0.0017687976360321045,
0.9689143896102905
] |
[
0.0017450300510972738,
0.9974599480628967,
0.000422302313381806,
0.0003727202711161226
] |
en
| 0.999996 |
BF: Prepared and edited the manuscript as well as the figures und performed echocardiographic evaluations; HKM: Drafted the manuscript and participated in the design of the study esp. immunological aspects; SO: Carried out TNF-α evaluations, recruited and coordinated the clinical management of the patients; SP: Carried out expression pattern analysis, recruited and coordinated the clinical management of the patient; IL: Participated in the study design; US: Reviewed the manuscript according to study design and immunological aspects PW and TG: Reviewed the manuscript and edited the figures; RZ: Participated in the development of LK565, the study design and coordination. All authors read and approved the final manuscript.
|
15357870_p24
|
15357870
|
Authors' contributions
| 0.805652 |
other
|
Other
|
[
0.2872425615787506,
0.00538927735760808,
0.7073681950569153
] |
[
0.006804967764765024,
0.9905850291252136,
0.0017068305751308799,
0.0009031407535076141
] |
en
| 0.999998 |
Several computational approaches to the problem of predicting cis-regulatory modules ('CRM's) have been reported recently. Berman et al . , Markstein et al . and Halfon et al . predicted CRM's involved in body patterning in the fly, and experimentally verified their predictions. The underlying principle in these algorithms was to detect dense clusters of binding sites, as determined by matches (above some threshold) to catalogued transcription factor weight matrices. The algorithm of Rajewsky et al . , called Ahab, avoided the use of thresholds on weight matrix matches by a probabilistic modeling of CRM's. Ahab predictions within the segmentation gene network were subjected to extensive experimental validation, with excellent overall success (Schroeder et al . ). Most predicted CRM's, when placed upstream of a reporter gene, faithfully reproduce one or more aspects of the endogenous gene expression pattern. Moreover, an analysis of binding site composition over the entire set of validated modules reveals that Ahab's prediction of binding sites correlates well with expression patterns produced by the modules and suggests basic rules governing module composition.
|
15357878_p0
|
15357878
|
Background
| 4.350582 |
biomedical
|
Study
|
[
0.9990098476409912,
0.000437952927313745,
0.0005521785351447761
] |
[
0.9435638189315796,
0.0006237648776732385,
0.05554768443107605,
0.00026470221928320825
] |
en
| 0.999998 |
The Stubb algorithm (Sinha et al . ) extended Ahab's approach by incorporating the use of two-species sequence information. Stubb also allows the option of scoring positional correlations between binding sites, but this option was not exercised in this study. For each sequence window analyzed, Stubb first computes the homologous sequence in the second species and aligns them using LAGAN (Brudno et al . ). The sequence is then partitioned into "blocks" (contiguous ungapped aligned regions of high percent identity) and non-blocks (sequence fragments between consecutive blocks, in either species). Putative binding sites in blocks are scored under an assumption of common evolutionary descent, using a probabilistic model of binding site evolution. Thus a "weak" site that is well conserved will score higher, while a "strong" site that is poorly conserved will have its score down-weighted. The score of the sequence window includes contributions from binding sites in blocks as well as in non-blocks. Stubb is implemented so that it can be run either on single species or two species data. In the single species mode, it is practically identical to the Ahab program. The Stubb software is available for download from
|
15357878_p1
|
15357878
|
Background
| 4.132615 |
biomedical
|
Study
|
[
0.9988788962364197,
0.0001929653953993693,
0.0009280433296225965
] |
[
0.9861363172531128,
0.011755876243114471,
0.0019396160496398807,
0.00016828547813929617
] |
en
| 0.999997 |
In this paper, we present evidence that the exploitation of cross-species comparison (between D. melanogaster and D. pseudoobscura ) using Stubb can lead to a significant improvement in the accuracy of genome-wide CRM prediction. To our knowledge, this is the first direct evaluation of the effect of cross-species comparison on CRM prediction on a genome-wide scale. Another important contribution of this paper is to present a benchmark for evaluating genome-wide CRM prediction tools, collected from the BDGP database and the literature, and curated by manual inspection of several hundred expression patterns. Using the same benchmark, we evaluate the effect of varying how background sequence information is incorporated in the algorithm, since this is the only tunable parameter in the Stubb program, other than the module length. We are thus able to suggest the optimal parameter settings for genome-wide CRM prediction using Stubb. Finally, we report all genome-wide predictions for cis-regulatory modules involved in anterior-posterior patterning in the early fly embryo, using both single-species and two-species Stubb, many of which make a strong case for experimental validation.
|
15357878_p2
|
15357878
|
Background
| 4.212096 |
biomedical
|
Study
|
[
0.9993414282798767,
0.00037906598299741745,
0.0002794408646877855
] |
[
0.999150276184082,
0.000189950704225339,
0.0005788811831735075,
0.0000809582561487332
] |
en
| 0.999995 |
The transcription control paradigm we use as our test system is the segmentation of the anterior-posterior (ap) axis during early Drosophila embryogenesis, which has long been one of the preferred arenas for studying transcription control in vivo . The segmentation genes form a hierarchical network that, in a process of stepwise refinement, translates broad, overlapping expression gradients into periodic patterns of 14 discrete stripes, which prefigure the 14 segments of the larva (for reviews see St Johnston & Nusslein-Volhard ; Rivera-Pomar & Jackle ; Furriols & Casanova ). The maternal factors form gradients stretching along the entire ap axis of the embryo, the zygotic "gap" factors are expressed in one or more broad slightly overlapping domains; together they generate the 7-stripe patterns of the pair-rule genes; finally, the segment-polarity genes are expressed in 14 stripes. The regulation within the segmentation gene hierarchy is almost entirely transcriptional, and most of the participating genes are transcription factors themselves, activating (in the case of the maternal factors) or repressing (most gap factors) the transcription of genes at the same level or below. In most cases, the relevant binding sites are clustered within a small interval of 0.5–1 kb; these CRM's typically contain binding sites for multiple transcription factors and multiple binding sites for each factor. The clustering and the combinatorial and redundant nature of the input facilitate the computational search for segmentation control elements. Since the expression patterns of the segmentation genes are typically complex, their control regions often contain multiple separate CRM's controlling different aspects of the pattern.
|
15357878_p3
|
15357878
|
Segmentation gene network
| 4.539858 |
biomedical
|
Study
|
[
0.9992091059684753,
0.00041429608245380223,
0.00037663584225811064
] |
[
0.9913033246994019,
0.0006231614970602095,
0.007891161367297173,
0.00018234421440865844
] |
en
| 0.999997 |
The segmentation paradigm has been used as a test system for the computational detection of CRMs by us and others (Rajewsky et al . , Schroeder et al . , Berman et al . , Grad et al . ). Here, as before (Schroeder et al . ), we use the maternal and zygotic gap factors Bicoid, Hunchback, Caudal, Knirps, Krüppel, Giant, Tailless, Dstat, and the TorRE binding factor as input to Stubb. The binding site specificity of each factor is characterized by a position weight matrix that is based on a collection of experimentally verified binding sites.
|
15357878_p4
|
15357878
|
Segmentation gene network
| 4.089972 |
biomedical
|
Study
|
[
0.9994342923164368,
0.00014518582611344755,
0.00042048306204378605
] |
[
0.9992710947990417,
0.0003997068270109594,
0.0002853537444025278,
0.000043942040065303445
] |
en
| 0.999998 |
The complete genomes of two fruitflies, D. melanogaster and D. pseudobscura have been sequenced, and Stubb was used to predict CRM's in the D. melanogaster genome. This was done in two modes – (i) STUBBSS, where Stubb is run on D. melanogaster genomic sequence alone, and (ii) STUBBMS, where Stubb uses orthologous sequence data from D. pseudobscura to help predict CRM's in D. melanogaster . For each mode of execution, we obtain a separate list of predicted CRM's, sorted in order of confidence in the prediction. The ideal test for our purpose would be to compare the accuracy of these two sorted lists. However, the set of experimentally verified CRM's involved in this system is sparse compared to the size of the system – roughly 50 CRM's are known (including the 15 new modules from Schroeder et al . ), while the number of target genes is several hundreds, by our estimate. Hence, direct evaluation of the success-rate of predictions is not feasible, and we use an alternative source of information to evaluate predictions, as described next.
|
15357878_p5
|
15357878
|
Evaluation methodology
| 4.143168 |
biomedical
|
Study
|
[
0.9993485808372498,
0.00019568904826883227,
0.0004557004722300917
] |
[
0.9993314743041992,
0.0003008713247254491,
0.00032103454577736557,
0.000046588244003942236
] |
en
| 0.999998 |
A functional CRM directs the expression of a gene, by definition, and typically this gene is located in close proximity to the CRM. Hence, we may map the list of predicted CRM's to a list of predicted blastoderm-patterned genes – for each CRM predicted by Stubb, the nearest gene is identified, and if this gene is less than a threshold distance of 20 Kbp away, it is predicted to be a blastoderm-patterned gene. The resulting list of predicted "patterned genes" may now be evaluated for accuracy. (Any duplicates in the list are removed before evaluation.) The Berkeley Drosophila Genome Project (BDGP) has catalogued the expression patterns of a large number of genes in D. melanogaster , at various stages of development. We considered such a catalogue of 2167 genes, obtained from BDGP and from the literature. (See Test Genes [ Additional File 1 ].) Visual inspection of the expression patterns of these genes revealed that 286 of them can be classified as having patterned expression along the anterior-posterior axis. (See Materials and Methods; also Patterned Genes [ Additional File 2 ].) Hence, our benchmark is the entire set of 2167 genes, the "positive" set is the 286 ap-patterned genes, the remaining 1881 forming the "negative" set. This enables us to evaluate the accuracy of lists of patterned genes predicted by STUBBSS and STUBBMS, and compare their performance.
|
15357878_p6
|
15357878
|
Evaluation methodology
| 4.179075 |
biomedical
|
Study
|
[
0.9994329810142517,
0.00027670382405631244,
0.0002903633576352149
] |
[
0.9993621706962585,
0.00026321475161239505,
0.0003210909490007907,
0.000053619733080267906
] |
en
| 0.999997 |
We note that some accuracy is lost in the translation of a list of predicted CRM's to the predicted genes it is mapped to, as per the mapping defined above. For instance, it is known that CRM's may control a gene located at large distances, i.e., further than the distance threshold of 20 Kb used in the mapping procedure. Also, it is possible that a CRM is located close to two genes, and directs the expression of both genes, or only of the farther gene, being somehow insulated from the nearer one. To address these concerns, we repeat our evaluation with a slightly different mapping from the one described above. A caveat that remains is that there may be genomic sequences that are functional, in the sense that they are capable of directing a specific blastoderm pattern in reporter gene constructs, but whose activity is 'silenced' in native genomic context and does not translate to patterning of any gene. Also, the CRM may direct expression of the gene only at post-blastodermal stages, so that the gene is not included in the "positive" test set of blastoderm patterned genes. Conversely, it may also happen that a predicted CRM lies close to a patterned gene, thereby being counted as a true positive, but the predicted CRM is not the sequence responsible for the gene's regulation. We assume that such effects are not biased against either algorithm.
|
15357878_p7
|
15357878
|
Evaluation methodology
| 4.176449 |
biomedical
|
Study
|
[
0.9994269609451294,
0.00023971458722371608,
0.00033335553598590195
] |
[
0.9985687732696533,
0.0005824482650496066,
0.0007829268579371274,
0.0000658220233162865
] |
en
| 0.999996 |
Figure 1a shows the results of our evaluation procedure on STUBBSS and STUBBMS. These results are for the best choice of parameters for each algorithm – local, 1 st order background for STUBBSS and global, 2 nd order background for STUBBMS. (The meanings of these parameter values are explained later in this section.) The x-axis is the number of unique genes that are predicted by the algorithm (by progressively decreasing its score threshold) and are in the set of 2167 genes with expression information. On the y-axis we plot how many of those predicted genes are in the "positive" set (i.e., have an ap blastoderm pattern.) Thus, the y-axis is the specificity of the algorithm. We observe that STUBBMS performs significantly better than STUBBSS. For instance, to predict 100 genes correctly, STUBBSS has to make 343 predictions, while STUBBMS only has to make 267 predictions. Figure 1b plots the difference in the number of correct predictions as a fraction of the number of correct STUBBSS predictions, i.e., the percentage change in specificity for the same number of predictions made by either algorithm. We find a typical improvement of over 20%, even when over 300 overall predictions are made by each algorithm. Figure 1c shows the progression of each algorithm's prediction specificity in a moving window of 50 predictions. We find that STUBBMS has a significantly higher hit rate for the first ~120 predictions, after which both algorithms perform comparably. Even for the lower ranked predictions (i.e., those below rank 120), we find a specificity of 20 – 35% with STUBBMS, which is roughly twice the random expectation of 13% based on 286 positives in 2167 genes.
|
15357878_p8
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 4.184469 |
biomedical
|
Study
|
[
0.9985083937644958,
0.00042011498589999974,
0.0010715511161834002
] |
[
0.9992744326591492,
0.00024734987528063357,
0.0004271361685823649,
0.00005101170972920954
] |
en
| 0.999996 |
In order to further scrutinize the difference in predictions made by the two modes of Stubb, we focused on the points where their difference is most pronounced. Thus, in the top 102 unique gene predictions (for which we have information), STUBBSS reports 39 positives, while STUBBMS scores 61 hits, an improvement of over 56%. In comparison, the random expectation is ~13.5 hits. Thus the predictions of both STUBBSS and STUBBMS are significantly enriched in patterned genes (P < 10 -12 and <10 -37 respectively, Binomial Proportions test). Further examination of the top 102 gene predictions made by each algorithm revealed that 24 true positives are common to both lists. STUBBMS reports 37 true positives not discovered by STUBBSS, while the latter reports 15 true positives not found by the former. Similar results are seen for the top 311 predictions : 70 correct predictions were common to both algorithms, 42 were predicted by STUBBMS only, and 21 by STUBBSS only. Thus there is substantial exclusivity in the sets of true positives of each algorithm.
|
15357878_p9
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 4.153919 |
biomedical
|
Study
|
[
0.9992169141769409,
0.00030194345163181424,
0.00048116318066604435
] |
[
0.9991570711135864,
0.0002817594795487821,
0.0005059009417891502,
0.00005532095019589178
] |
en
| 0.999997 |
We next examined separately the following three sets of genes: (i) INTERSECTION (predicted by both algorithms in the top 311) (ii) MS-ONLY (predicted only by STUBBMS) and (iii) SS-ONLY (predicted only by STUBBSS). Table 1 shows the break-down of these sets in terms of the strength of expression of their member genes. Overall, 124 of the 286 patterned genes, i.e., about 43%, are strongly expressed. We find in Table 1 that the sets INTERSECTION and MS-ONLY have more strongly expressed genes than weak and intermediate ones, and the opposite trend is seen in the set SS-ONLY.
|
15357878_p10
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 3.957641 |
biomedical
|
Study
|
[
0.9988186955451965,
0.0002037492668023333,
0.0009775133803486824
] |
[
0.999417781829834,
0.00031589544960297644,
0.00022489321418106556,
0.00004134913979214616
] |
en
| 0.999998 |
One possible strategy that uses two-species sequence is to make predictions using STUBBSS on each of the two genomes separately and then intersect the respective lists. We found this strategy to be very restrictive – for instance, with a particular score threshold, STUBBSS predicts 205 unique genes in D. melanogaster , but intersecting these predictions with a similar number of top predictions in D. pseudoobscura gives only 68 unique genes, 33 of which are patterned. Of the top 68 predictions made in D. melanogaster alone, 29 are patterned. Thus the "intersection" strategy yields only a modest improvement over the single-species search, and does so at the price of significantly reducing the total number of predictions. Similar results were obtained when intersecting modules instead of gene predictions.
|
15357878_p11
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 4.111372 |
biomedical
|
Study
|
[
0.9991658926010132,
0.00018363981507718563,
0.0006504068151116371
] |
[
0.9989820122718811,
0.0006932738469913602,
0.0002744256053119898,
0.00005029303429182619
] |
en
| 0.999996 |
We have noted above that the evaluation method is influenced by the way we map the predicted CRM's to predicted genes. To offset potential biases induced by this mapping, we repeated our analysis with a slightly different evaluation procedure, borrowing from the approach of Grad et al . . We now traverse the sorted list of CRM's and count a CRM as a prediction if either of its two flanking genes has expression information. Furthermore, we designate a prediction to be "correct" if either of the two flanking genes has a blastoderm pattern. The assumption, as in Grad et al . , is that any predicted CRM near a blastoderm-patterned gene is a functional CRM responsible for some aspect of the pattern. Also, we are now counting modules rather than genes, i.e. we are allowing for multiple hits to the same gene. Figure 2a plots the results of STUBBSS and STUBBMS as per this new method of counting predictions and hits. We again notice a significant improvement in STUBBMS. For instance, in the top 300 CRM predictions for which a neighboring gene has expression information, STUBBMS makes 160 correct predictions while STUBBSS scores 121 hits. The gap between STUBBSS and STUBBMS increases as more predictions are considered, so that the improvement consistently stays above 20%, as seen in Figure 2b . For the remainder of this section, our evaluation method will use the more stringent mapping described earlier, wherein the nearest gene is predicted. Since our test data is in the form of lists of genes, we adhere to the evaluation strategy that counts genes. It is clear from Figures 1 and 2 that counting modules rather than genes improves the prediction accuracy, due to multiple CRM predictions for some blastoderm-patterned genes.
|
15357878_p12
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 4.169852 |
biomedical
|
Study
|
[
0.9991533756256104,
0.00032675539841875434,
0.0005198511644266546
] |
[
0.9994118213653564,
0.00016823892656248063,
0.0003657928027678281,
0.000054169642680790275
] |
en
| 0.999998 |
The default mapping from CRM's to genes used in our evaluations predicts a gene to be patterned only if its proximal end is less than 20 Kb from the CRM. Schroeder et al . studied the range of locations of experimentally verified CRM's relative to the gene. They found that while there is a clustering of CRM's within the proximal 5 Kb region upstream, downstream or intronic of a gene, it is not unusual to have CRM's more than 10 Kb away from the regulated gene. Nelson et al . observe that for D. melanogaster, the intergenic space on either side of a gene has a mean of 2 Kb – 10 Kb, depending on the complexity of the gene's function. We repeated our evaluation with different values of the distance threshold, and found that lower thresholds (5 Kb, 10 Kb) decrease the recovery rate, while higher thresholds (50 Kb) do not affect performance. (Data not shown.)
|
15357878_p13
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 4.112138 |
biomedical
|
Study
|
[
0.9995032548904419,
0.00018166034715250134,
0.0003150544362142682
] |
[
0.9992305040359497,
0.0002427288272883743,
0.00047246264875866473,
0.00005435302227851935
] |
en
| 0.999999 |
Genes in the segmentation hierarchy often have multiple aspects to their expression pattern, with more than one CRM regulating them. We therefore measured how the Stubb predictions fare if we required that each predicted gene be evidenced by at least two predicted CRM's. This heuristic improves the performance of STUBBSS more prominently than that of STUBBMS, though much fewer predictions are made by either algorithm. While 342 unique gene predictions were made by STUBBSS , we now observe that only 105 predictions are made using the same score threshold and the new way of counting predictions. Thus, it appears that STUBBSS performance is open to considerable improvement in the top ~100 predictions, by using either the multiple CRM restriction or the second species' sequence data. The two-species strategy however is able to increase specificity without loss of sensitivity.
|
15357878_p14
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 4.09479 |
biomedical
|
Study
|
[
0.999329686164856,
0.00021734180336352438,
0.0004529987054411322
] |
[
0.9993352293968201,
0.00028801895678043365,
0.00032728401129134,
0.00004942672967445105
] |
en
| 0.999998 |
We have, in all tests reported in this paper, used as input a set of 2167 genes whose expression patterns are available either from BDGP or from the literature. BDGP has a supplementary list of 2065 genes for which only textual annotation has been made public, since these genes have been found to be either (i) ubiquitously expressed at all developmental stages, (ii) not expressed at any stage, or (iii) only maternally expressed. (See Additional Genes [ Additional File 3 ].) Inclusion of these supplementary genes in our data set would approximately halve the overall fraction of patterned genes. When we examine the performance curves of STUBBSS and STUBBMS for this pattern-diluted data set , we find that STUBBMS shows an improvement over STUBBSS similar in proportion to that in the default data set, even though the prediction specificity of both programs suffers a drop , typically in the range of 10–30%. Note, however, that this is substantially lower than the 50% drop one would expect by chance, given that the total number of genes has almost doubled, while the number of patterned genes remains constant.
|
15357878_p15
|
15357878
|
STUBBMS performs significantly better than STUBBSS
| 4.103528 |
biomedical
|
Study
|
[
0.9993682503700256,
0.00024623697390779853,
0.00038549714372493327
] |
[
0.999462902545929,
0.00018786630243994296,
0.00030244782101362944,
0.000046870598453097045
] |
en
| 0.999997 |
Our annotations of the blastoderm patterned genes also include whether the gene expression is strong, weak or of intermediate strength; if it has a dorsal-ventral (dv) modulation in addition to the primary anterior-posterior pattern; and if the gene belongs to a "core" set of 48 genes that have been shown experimentally to be required for the segmentation of the embryo (Schroeder et al . ). We were therefore able to examine the characteristics of the genes correctly predicted by Stubb, along these axes of information. The top 135 correct (gene) predictions made by STUBBMS were examined progressively, 20 predictions at a time. (That is, the correct predictions ranked I to I+19 were examined, with I being incremented in steps.) In each step, we computed the fraction of the 20 genes that belonged to the following three non-exclusive categories: (i) genes with dv (in addition to ap) modulation, (ii) genes with strong expression pattern, and (iii) genes in the "core" set of 48 genes. These values are reported in Figure 5 . We find that
|
15357878_p16
|
15357878
|
Characteristics of genes predicted by STUBBMS
| 4.125054 |
biomedical
|
Study
|
[
0.9992413520812988,
0.0003104795760009438,
0.00044820079347118735
] |
[
0.9993595480918884,
0.00016807745851110667,
0.00041829689871519804,
0.00005407436401583254
] |
en
| 0.999995 |
1. Genes with dorsal-ventral aspects to their blastoderm pattern are more frequent at lower ranks of prediction; i.e., the top predictions are enriched in genes with anterior-posterior patterns only.
|
15357878_p17
|
15357878
|
Characteristics of genes predicted by STUBBMS
| 2.940893 |
biomedical
|
Study
|
[
0.9870004057884216,
0.0007109309663064778,
0.012288760393857956
] |
[
0.567894458770752,
0.42650315165519714,
0.0045721204951405525,
0.0010301885195076466
] |
en
| 0.999994 |
2. Core genes are predominantly found in the top predictions.
|
15357878_p18
|
15357878
|
Characteristics of genes predicted by STUBBMS
| 2.13834 |
biomedical
|
Other
|
[
0.9543871879577637,
0.002969332505017519,
0.04264348745346069
] |
[
0.32038968801498413,
0.666171133518219,
0.010943500325083733,
0.0024956900160759687
] |
en
| 0.999997 |
3. Genes found at higher ranks are somewhat more likely to be strongly expressed.
|
15357878_p19
|
15357878
|
Characteristics of genes predicted by STUBBMS
| 2.203908 |
biomedical
|
Other
|
[
0.9560047388076782,
0.001558836316689849,
0.04243648424744606
] |
[
0.424435019493103,
0.5647950768470764,
0.00926908478140831,
0.0015007884940132499
] |
en
| 0.999996 |
The first two observations imply that the genes more directly involved in the ap axis formation are recovered at better ranks, and that the lower rank genome-wide predictions are richer in derivative patterns characteristic of genes with more complex regulatory inputs (pair-rule factors, dv factors etc.). The same trends were found for the correct predictions made by STUBBSS. (Data not shown.)
|
15357878_p20
|
15357878
|
Characteristics of genes predicted by STUBBMS
| 3.526307 |
biomedical
|
Study
|
[
0.9983920454978943,
0.000223176772124134,
0.0013848006492480636
] |
[
0.9968980550765991,
0.0024087654892355204,
0.0005932313506491482,
0.00009988240344682708
] |
en
| 0.999996 |
We next evaluate the effect of varying how background sequence information is incorporated in the Stubb algorithm. This is the only configurable aspect of the program, other than the module length. (In a separate test, we ran Stubb with a module length of 700 instead of the default value of 500, and found no significant difference in the prediction specificity curve.) One important parameter is the "Markov order" of background. A value of k for this parameter means that local correlations are assumed to be present at the level of ( k+1 )-mers, i.e., the random probability of seeing a particular base at a position depends on the bases seen at the previous k positions. (For readers familiar with the studies of Rajewsky et al . and Schroeder et al . , "background k " in those studies is the same as a (k-1) th order background in the terminology of this paper.) We vary this parameter to take the values k = 1 and k = 2 , in different runs. The other parameter is the actual sequence used by Stubb to measure background nucleotide frequencies. Here the two options are (i) to use the current sequence window as background, or (ii) to use a pre-specified sequence (or collection of sequences) as background. We call these two the "local" and "global" background models respectively. For the "global" model, we input into Stubb 150 Kb of sequence from non-coding regions of the D. melanogaster genome, collected from the five chromosome arms 2L, 2R, 3L, 3R, and X.
|
15357878_p21
|
15357878
|
Optimal parameter settings for Stubb
| 4.145642 |
biomedical
|
Study
|
[
0.9988825917243958,
0.00028710844344459474,
0.0008303193026222289
] |
[
0.9994388222694397,
0.000265393900917843,
0.0002531647915020585,
0.00004263027585693635
] |
en
| 0.999997 |
Figure 6 plots the specificity curves for each combination of parameter values tested. Figure 6a reports on different variants of STUBBSS, while Figure 6b plots the performance of STUBBMS. We find that STUBBSS performs best with a local, 1 st order background, though the other parameter values produce only slightly different results. On the other hand, the effect of background parameters on two-species Stubb is more pronounced, with the best choice being a global, 2 nd order background. Using a global 1 st background order gives almost identical results (data not shown), hence we infer that a global background is the optimal choice for STUBBMS.
|
15357878_p22
|
15357878
|
Optimal parameter settings for Stubb
| 3.442197 |
biomedical
|
Study
|
[
0.9532172083854675,
0.0006421376601792872,
0.04614066332578659
] |
[
0.9986655712127686,
0.0010312016820535064,
0.00024234046577475965,
0.00006084956839913502
] |
en
| 0.999996 |
As mentioned earlier, the STUBBSS program implements the same class of algorithm as the Ahab algorithm of Rajewsky et al . , with some technical differences, and therefore the two programs should produce similar results. We sought to verify this claim by running the Ahab program (with 1 st and 2 nd order Markov backgrounds), and comparing its performance to that of Stubb. (Ahab can only be run in the local background mode.) Figure 7a shows that there is not a significant difference between Stubb and Ahab CRM predictions.
|
15357878_p23
|
15357878
|
Optimal parameter settings for Stubb
| 2.680422 |
biomedical
|
Study
|
[
0.8160519003868103,
0.0008329763659276068,
0.18311503529548645
] |
[
0.9832533001899719,
0.015989521518349648,
0.0005401048110798001,
0.00021703512175008655
] |
en
| 0.999998 |
All the above runs were on genomic sequence with tandem repeats masked by the Tandem Repeats Finder program of Benson . We have found that this heuristic improves genome-wide CRM prediction by Stubb. To substantiate this claim, we ran STUBBSS and STUBBMS on raw (unmasked) genomic sequence. Figure 7b plots the results. We find that both STUBBSS and STUBBMS perform better on masked data than on unmasked data. However, when Stubb is used to analyze shorter sequences (such as the upstream and downstream regions of a gene of interest), we have found unmasked sequence to be more useful, since false positives are less of a concern.
|
15357878_p24
|
15357878
|
Optimal parameter settings for Stubb
| 4.071527 |
biomedical
|
Study
|
[
0.9993466734886169,
0.00023268941731657833,
0.0004206368757877499
] |
[
0.9994857311248779,
0.00022290620836429298,
0.00023962149862200022,
0.00005176839113119058
] |
en
| 0.999997 |
The Stubb program is an extension of Ahab, with the important feature that it can handle two-species data within its probabilistic framework. The two programs differ in their underlying optimization method, with Stubb using an Expectation-Maximization approach in contrast to Ahab's conjugate gradient method. Performance evaluation of the two programs shows little difference between them, implying that the algorithm is robust to the actual optimization method used. Another technical difference between Ahab and Stubb is in the manner that orientation of binding sites is treated. While Stubb assumes a uniform prior on the orientation of a binding site, Ahab picks the best orientation for each site, with the caveat that probabilities are not strictly normalized.
|
15357878_p25
|
15357878
|
Discussion
| 3.958625 |
biomedical
|
Study
|
[
0.9918204545974731,
0.00032624040613882244,
0.007853366434574127
] |
[
0.7719234824180603,
0.22149558365345,
0.006194495130330324,
0.00038637674879282713
] |
en
| 0.999996 |
An important component of Stubb is the alignment step where the two species are aligned (using LAGAN) and blocks of high sequence similarity are extracted. (See Methods.) The parameters used in LAGAN runs were obtained from Emberly et al . , who derived the alignment parameters that maximize the overlap between experimentally verified binding sites and blocks of sequence conservation. They also studied the effect of changing the alignment algorithm (LAGAN from Brudno et al . versus SMASH from Zavolan et al . ) for CRM's in the two fly species, and found no significant difference. Finally, the similarity thresholds we use for defining conserved blocks (10 bp or longer, with >70% identity) were obtained by trying a broad range of values, and choosing those that produced the best results, as per our genome-wide evaluation.
|
15357878_p26
|
15357878
|
Discussion
| 4.117499 |
biomedical
|
Study
|
[
0.9993855953216553,
0.00019341029110364616,
0.0004210303595755249
] |
[
0.999451220035553,
0.0002669231907930225,
0.00024027407926041633,
0.00004150156382820569
] |
en
| 0.999997 |
Tandem repeat masking is a common pre-processing step for many sequence analysis applications involving binding sites. These repeats are short locally duplicated sequences, that may or may not be related to binding sites. It is not clear a priori how tandem repeats should affect module detection – repeats similar to binding sites of the system may improve sensitivity when they occur in CRM's; but if repeats resembling binding sites occur by chance in non-functional regions, prediction specificity may suffer. The occurrence of tandem repeats marks statistical deviation from Stubb's probabilistic model of sequence generation. In our tests, we found that repeats distract the algorithm more than they help, as manifested in better performance on repeat-masked sequence. This may be because two of the weight matrices in our collection (Hunchback and Caudal) resemble a poly-T stretch. Therefore, the poly-A or poly-T tandem repeats that occur promiscuously in the genome may be confused with sites of these two weight matrices.
|
15357878_p27
|
15357878
|
Discussion
| 4.17401 |
biomedical
|
Study
|
[
0.9994096755981445,
0.00019480906485114247,
0.0003955028369091451
] |
[
0.9992189407348633,
0.0003711599565576762,
0.000351844122633338,
0.0000579563420615159
] |
en
| 0.999997 |
A recently published tool for genome-wide CRM prediction, called PFR-Searcher (Grad et al . ), first identifies "phylogenetically footprinted regions" or "PFR"s, that are sequences conserved between the two fly species, and then searches for a subset of these that are most similar in content to an input set of promoters. Their approach differs from Stubb in the nature of prior information input to the algorithm. While Stubb uses an input set of weight matrices, the training data for PFR-Searcher is a set of CRM's which, in their approach, is itself provided by a similarity search among PFR's of co-regulated genes. PFR-Searcher therefore has the advantage of not requiring knowledge of the transcription factor weight matrices relevant to the system. However, its ability to predict the binding site composition of potential CRM's is therefore more limited as compared to Stubb. (The Stubb program computes an average "parse" of the predicted module into its constituent binding sites for various transcription factors.) Grad et al . report an evaluation of their algorithm on a test system very similar to ours, but with enough minor differences to make a direct comparison of performance impossible. For instance, the entire list of CRM's predicted in their evaluation corresponds, as per our CRM → gene mapping, to a set of only 46 unique genes, of which 31 are patterned. Twenty of these 31 correct predictions are also found in the top 46 gene predictions of STUBBMS, indicating a good degree of overlap between the two methods, at least in their highest ranked predictions. A fair and comprehensive comparison of the predictive power of these two algorithms is an interesting topic for future work, and it will be even more interesting to run STUBBMS only on PFR's detected by their criteria.
|
15357878_p28
|
15357878
|
Discussion
| 4.355996 |
biomedical
|
Study
|
[
0.9992372989654541,
0.0003116102598141879,
0.0004510418511927128
] |
[
0.9821020364761353,
0.0006538432789966464,
0.01709158718585968,
0.0001524208055343479
] |
en
| 0.999998 |
Regarding the recovery of patterned genes by Stubb, several observations can be made. Of the 286 genes with ap patterns, we recover roughly half at a score cut-off of 10, using STUBBMS. Why is the other half not found? While it is obvious that lowering the cut-off will detect more patterned genes, there are other reasons why a patterned gene may be missed by Stubb. Some genes are likely to be lost due to the distance filter we have imposed (CRM to nearest gene <20 kb), since the regulatory regions of some genes (e.g., homeotic genes) are likely to be larger than that. More importantly, most of the patterned genes that are not part of the core transcriptional machinery have derivative patterns that reflect a more complex input (binding sites for pair rule factors, d-v factors etc.) and thus will only be recovered to the extent their input has a solid maternal/gap component. Conversely, there are at least two reasons for reporting false positives (roughly two thirds at a score cutoff of 10). The presence of an insulator could prevent the interaction between a CRM and its nearest basal promoter. More likely is a scenario where the predicted CRM's do drive expression but at post-blastoderm stages. All gap factors are active in multiple tissues in later development and therefore CRM's with dominant or exclusive gap input may well be active in these later contexts. These caveats affect all current CRM detection algorithms, and accounting for such additional axes of information as genomic context and module composition rules will be a difficult but important challenge for the future.
|
15357878_p29
|
15357878
|
Discussion
| 4.269666 |
biomedical
|
Study
|
[
0.9993684887886047,
0.0003105664800386876,
0.00032097421353682876
] |
[
0.9983643889427185,
0.0004288216878194362,
0.0011255790013819933,
0.00008115483069559559
] |
en
| 0.999998 |
A very interesting observation comes from the analysis in Table 1 : Genes predicted by STUBBSS only, and not by STUBBMS, have weak or intermediate expression pattern more often than strong expression. This means that the CRM's that are not well-conserved between the two species (and hence not picked up by STUBBMS) typically correspond to weakly expressed genes. This ties in with previous studies (e.g., Domazet-Loso & Tautz ) that found fast evolving genes in Drosophila to be expressed relatively weakly.
|
15357878_p30
|
15357878
|
Discussion
| 3.945091 |
biomedical
|
Study
|
[
0.9989078044891357,
0.00018466281471773982,
0.0009075009147636592
] |
[
0.9989495873451233,
0.0006289834273047745,
0.0003663918760139495,
0.000055000280553940684
] |
en
| 0.999997 |
The Stubb program not only predicts cis-regulatory modules genome-wide, it additionally outputs the binding site profile of each predicted CRM, i.e., the locations and probabilities of binding sites in the CRM. Schroeder et al use the corresponding feature in Ahab for a systematic analysis of the composition of all known or validated segmentation CRMs. The use of STUBBMS improves such binding site predictions. It is easy to adapt the program to take as input orthologous CRM's from the two species, and highlight the changes in terms of their binding site compositions. This leads to a powerful bioinformatic tool to predict regulatory changes between the two fly species. We can thus obtain hypotheses about changes in expression patterns, which can be verified experimentally. We have examined a representative collection of CRM's, and experimentally verified several of the changes predicted by Stubb, thereby building a catalogue of the different modes of cis-regulatory evolution. The results of this study will be reported in the near future.
|
15357878_p31
|
15357878
|
Discussion
| 4.160652 |
biomedical
|
Study
|
[
0.9995402097702026,
0.0001804447383619845,
0.00027940821019001305
] |
[
0.998534083366394,
0.0007471919525414705,
0.0006494707195088267,
0.0000692820904077962
] |
en
| 0.999999 |
We have seen that the use of a second fly genome significantly improves genome-wide module prediction. Since STUBBMS uses a natural "two-species" extension of the algorithm of STUBBSS, this finding is largely a statement about the inherent potential of cross-species comparison as a paradigm for improving functional genomics. The STUBBMS program also has a natural extension to incorporate more than two genomes, and it will be very interesting to see how much of a difference a third genome makes. The genome of D. yakuba is expected to be sequenced soon, and since this species is closer to D. melanogaster, it may help better discriminate conserved regulatory modules.
|
15357878_p32
|
15357878
|
Conclusions
| 4.047606 |
biomedical
|
Study
|
[
0.9995052814483643,
0.0001156711732619442,
0.0003789830079767853
] |
[
0.9951000809669495,
0.003813098417595029,
0.0009860184509307146,
0.00010077078331960365
] |
en
| 0.999997 |
D. melanogaster sequences were obtained from Flybase Release 3. The analysis was limited to the five chromosome arms 2L, 2R, 3L, 3R, and X. D. pseudobscura contigs were obtained from . Based on Blast results, we created a mapping, called "CONTIGMAP", between regions of the D. melanogaster genome and D. pseudobscura contigs, each region typically being tens of Kb long. This mapping is many to many, i.e., different regions of D. melanogaster may map to the same contig, and the same (or overlapping) region in D. melanogaster may map to two or more D. pseudobscura contigs. For each entry (M, P) in CONTIGMAP, where M is the D. melanogaster region and P is the D. pseudobscura contig, the LAGAN alignment program (Brudno et al . ) was run, with parameters gap start = -6, gap extension = 0, match = 1, and mismatch = -2, and all contiguous ungapped blocks of alignment, with length 10 bp or more and 70% identity or more, were extracted. In cases where the same region in D. melanogaster was mapped to multiple contigs, the density of LAGAN blocks was then used to choose exactly one mapping contig.
|
15357878_p33
|
15357878
|
Alignment of D. melanogaster and D. pseudobscura
| 4.20825 |
biomedical
|
Study
|
[
0.9994238615036011,
0.00023671533563174307,
0.00033943381276912987
] |
[
0.9993212223052979,
0.00033010338665917516,
0.0002892649208661169,
0.000059459503972902894
] |
en
| 0.999996 |
Tandem repeats in the input sequences were masked with the Tandem Repeat Finder program of Benson , with parameter settings: (match = 2, mismatch = 5, indel = 5, match probability = 0.75, indel probability = 0.2, minimum score = 20, maximum period = 500). STUBBSS was run on the D. melanogaster genome with a sliding window of length 500 bp, in shifts of 50 bp. The input weight matrices for the maternal and gap transcription factors Bcd, Hb, Cad, Kni, Kr, Tll, Dstat and the torRE binding factor were obtained from Rajewsky et al . and Schroeder et al . . A weight matrix for the transcription factor Gt was constructed from known functional sites collected from the literature. STUBBMS was run on each entry (M, P) in CONTIGMAP, using a sliding window of length 500 bp on the D. melanogaster sequence M, in shifts of 50 bp. Thus, STUBBMS was not run on regions of D. melanogaster that are not aligned with some D. pseudoobscura contig. The weight matrices used were the same as in STUBBSS runs. The locations of the blocks computed in the alignment step (above) were input to STUBBMS, and the input value of the neutral mutation rate was 0.5, the value being chosen due to its better performance over alternatives tested.
|
15357878_p34
|
15357878
|
Stubb runs
| 4.188855 |
biomedical
|
Study
|
[
0.9994211196899414,
0.00025769422063603997,
0.0003211454313714057
] |
[
0.9994254112243652,
0.00023814108863007277,
0.00027567343204282224,
0.00006075021519791335
] |
en
| 0.999998 |
Subsets and Splits
SQL Console for rntc/test-pp-aa
The query retrieves a sample of documents that are clinical cases with an educational score above 3, providing limited analytical value.
Clinical Cases Sample
Returns a sample of 100 clinical case documents, providing a basic overview of the document type's content.