606 research outputs found
A Rapid and Quantitative Assay to Estimate Gene Transfer into Retrovirally Transduced Hematopoietic Stem/Progenitor Cells Using a 96-Well Format PCR and Fluorescent Detection System Universal for MMLV-Based Proviruses
Overview summary The polymerase chain reaction (PCR) analysis of colonies of clonogenic cells growing in methylcellulose medium is a routine procedure to estimate the frequency of retroviral transduction into hematopoietic stem/progenitor cells. This study describes a sensitive assay system that takes advantage of the standard 96-well format to expedite the processing of single methylcellulose colonies. Assay sensitivity is dependent on a PCR primer pair which amplifies a region of the ψ packaging sequence of all Moloney-based retroviruses tested. Using this primer pair, we present the optimized PCR conditions for the analysis of single colonies of clonogenic cells growing in methylcellulose medium as well as the conditions for a semiquantitative bulk PCR assay to estimate the transduction frequency immediately following the transduction protocol. This PCR primer pair, along with the capability for more rapid screening of hematopoietic stem/progenitor colonies, is especially useful for the laboratory that is screening a number of different retroviral constructions for their transduction efficiency.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63170/1/hum.1996.7.3-343.pd
Developing a Bayesian network model for understanding river catchment resilience under future change scenarios
Rofecoxib and cardiovascular adverse events in adjuvant treatment of colorectal cancer
Background
Selective cyclooxygenase inhibitors may retard the progression of cancer, but they
have enhanced thrombotic potential. We report on cardiovascular adverse events in
patients receiving rofecoxib to reduce rates of recurrence of colorectal cancer.
Methods
All serious adverse events that were cardiovascular thrombotic events were reviewed
in 2434 patients with stage II or III colorectal cancer participating in a randomized,
placebo-controlled trial of rofecoxib, 25 mg daily, started after potentially curative
tumor resection and chemotherapy or radiotherapy as indicated. The trial was terminated
prematurely owing to worldwide withdrawal of rofecoxib. To examine possible
persistent risks, we examined cardiovascular thrombotic events reported up to 24
months after the trial was closed.
Results
The median duration of active treatment was 7.4 months. The 1167 patients receiving
rofecoxib and the 1160 patients receiving placebo were well matched, with a median
follow-up period of 33.0 months (interquartile range, 27.6 to 40.1) and 33.4 months
(27.7 to 40.4), respectively. Of the 23 confirmed cardiovascular thrombotic events,
16 occurred in the rofecoxib group during or within 14 days after the treatment
period, with an estimated relative risk of 2.66 (from the Cox proportional-hazards
model; 95% confidence interval [CI], 1.03 to 6.86; P = 0.04). Analysis of the Antiplatelet
Trialists’ Collaboration end point (the combined incidence of death from
cardiovascular, hemorrhagic, and unknown causes; of nonfatal myocardial infarction;
and of nonfatal ischemic and hemorrhagic stroke) gave an unadjusted relative
risk of 1.60 (95% CI, 0.57 to 4.51; P = 0.37). Fourteen more cardiovascular thrombotic
events, six in the rofecoxib group, were reported within the 2 years after trial
closure, with an overall unadjusted relative risk of 1.50 (95% CI, 0.76 to 2.94;
P = 0.24). Four patients in the rofecoxib group and two in the placebo group died
from thrombotic causes during or within 14 days after the treatment period, and
during the follow-up period, one patient in the rofecoxib group and five patients in
the placebo group died from cardiovascular causes.
Conclusions
Rofecoxib therapy was associated with an increased frequency of adverse cardiovascular
events among patients with a median study treatment of 7.4 months’ duration.
(Current Controlled Trials number, ISRCTN98278138.
Mapping of mitral regurgitant defects by cardiovascular magnetic resonance in moderate or severe mitral regurgitation secondary to mitral valve prolapse
Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues
Introduction:
The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.
Methods:
SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.
Results:
Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.
Conclusion:
Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets
Rapid, -insensitive, dual-band quasi-adiabatic saturation transfer with optimal control for complete quantification of myocardial ATP flux
Purpose: Phosphorus saturation-transfer experiments can quantify metabolic
fluxes non-invasively. Typically, the forward flux through the creatine-kinase
reaction is investigated by observing the decrease in phosphocreatine (PCr)
after saturation of -ATP. The quantification of total ATP utilisation
is currently under-explored, as it requires simultaneous saturation of
inorganic phosphate (Pi) and PCr. This is challenging, as currently available
saturation pulses reduce the already-low -ATP signal present.
Methods: Using a hybrid optimal-control and Shinnar-Le-Roux method, a
quasi-adiabatic RF pulse was designed for the dual-saturation of PCr and Pi to
enable determination of total ATP utilisation. The pulses were evaluated in
Bloch equation simulations, compared with a conventional hard-cosine DANTE
saturation sequence, before application to perfused rat hearts at 11.7 Tesla.
Results: The quasi-adiabatic pulse was insensitive to a -fold variation
in , producing equivalent saturation with a 53% reduction in delivered
pulse power and a 33-fold reduction in spillover at the minimum effective
. This enabled the complete quantification of the synthesis and
degradation fluxes for ATP in 30-45 minutes in the perfused rat heart. While
the net synthesis flux ( mM/s, SEM) was not significantly different
from degradation flux ( mM/s, ) and both measures are
consistent with prior work, nonlinear error analysis highlights uncertainties
in the Pi-to-ATP measurement that may explain a trend suggesting a possible
imbalance.
Conclusion: This work demonstrates a novel quasi-adiabatic dual-saturation RF
pulse with significantly improved performance that can be used to measure ATP
turnover in the heart in vivo.Comment: 26 pages, Accepted at Magnetic Resonance in Medicine, 24/11/2020
[This version post reviews
Factors affecting consistency and accuracy in identifying modern macroperforate planktonic foraminifera
Planktonic foraminifera are widely used in biostratigraphic, palaeoceanographic and evolutionary studies, but the strength of many study conclusions could be weakened if taxonomic identifications are not reproducible by different workers. In this study, to assess the relative importance of a range of possible reasons for among-worker disagreement in identification, 100 specimens of 26 species of macroperforate planktonic foraminifera were selected from a core-top site in the subtropical Pacific Ocean. Twenty-three scientists at different career stages – including some with only a few days experience of planktonic foraminifera – were asked to identify each specimen to species level, and to indicate their confidence in each identification. The participants were provided with a species list and had access to additional reference materials. We use generalised linear mixed-effects models to test the relevance of three sets of factors in identification accuracy: participant-level characteristics (including experience), species-level characteristics (including a participant’s knowledge of the species) and specimen-level characteristics (size, confidence in identification). The 19 less experienced scientists achieve a median accuracy of 57 %, which rises to 75 % for specimens they are confident in. For the 4 most experienced participants, overall accuracy is 79 %, rising to 93 % when they are confident. To obtain maximum comparability and ease of analysis, everyone used a standard microscope with only 35× magnification, and each specimen was studied in isolation. Consequently, these data provide a lower limit for an estimate of consistency. Importantly, participants could largely predict whether their identifications were correct or incorrect: their own assessments of specimen-level confidence and of their previous knowledge of species concepts were the strongest predictors of accuracy
Synthesis of (+)-(R)-Tiruchanduramine
The absolute stereochemistry of the marine alkaloid (+)-(R)-tiruchanduramine was established via a convergent total synthesis in six steps and 15.5% overall yield from Fmoc-D-Dab(Boc)-OH
Pion-nucleus elastic scattering on 12C, 40Ca, 90Zr, and 208Pb at 400 and 500 MeV
Pion-nucleus elastic scattering at energies above the Delta(1232) resonance
is studied using both pi+ and pi- beams on 12C, 40Ca, 90Zr, and 208Pb. The
present data provide an opportunity to study the interaction of pions with
nuclei at energies where second-order corrections to impulse approximation
calculations should be small. The results are compared with other data sets at
similar energies, and with four different first-order impulse approximation
calculations. Significant disagreement exists between the calculations and the
data from this experiment
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