456 research outputs found
Sequence sensitivity of breathing dynamics in heteropolymer DNA
We study the fluctuation dynamics of localized denaturation bubbles in
heteropolymer DNA with a master equation and complementary stochastic
simulation based on novel DNA stability data. A significant dependence of
opening probability and waiting time between bubble events on the local DNA
sequence is revealed and quantified for a biological sequence of the T7
bacteriophage. Quantitative agreement with data from fluorescence correlation
spectroscopy (FCS) is demonstrated.Comment: 4 pages, 5 figures, to appear in Physical Review Letter
Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA
Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA
Bubble dynamics in DNA
The formation of local denaturation zones (bubbles) in double-stranded DNA is
an important example for conformational changes of biological macromolecules.
We study the dynamics of bubble formation in terms of a Fokker-Planck equation
for the probability density to find a bubble of size n base pairs at time t, on
the basis of the free energy in the Poland-Scheraga model. Characteristic
bubble closing and opening times can be determined from the corresponding first
passage time problem, and are sensitive to the specific parameters entering the
model. A multistate unzipping model with constant rates recently applied to DNA
breathing dynamics [G. Altan-Bonnet et al, Phys. Rev. Lett. 90, 138101 (2003)]
emerges as a limiting case.Comment: 9 pages, 2 figure
Local anesthetics worsen renal function after ischemia-reperfusion injury in rats
. Local anesthetics worsen renal function after ischemia-reperfusion injury in rats. Am J Physiol Renal Physiol 286: F111-F119, 2004. First published September 30, 2003 10.1152 10. /ajprenal.00108.2003ics are widely used during the perioperative period, even in patients with preexisting renal disease. However, local anesthestics have been shown to cause cell death in multiple cell lines, including human kidney proximal tubule cells. We questioned whether local anesthetics potentiate renal dysfunction after ischemia-reperfusion (I/R) injury in vivo. Rats were implanted with subcutaneous miniosmotic pumps that continuously delivered lidocaine (2 mg⅐kg Ϫ1 ⅐h Ϫ1 ), bupivacaine (0.4 mg⅐kg Ϫ1 ⅐h Ϫ1 ), tetracaine (1 mg⅐kg Ϫ1 ⅐h Ϫ1 ), or saline vehicle, and 6 h later the rats were subjected to 30 min of renal ischemia or to sham operation. Renal function was assessed by measurement of plasma creatinine at 24 and 48 h after renal I/R injury in the presence or absence of chronic infusions of local anesthetics and correlated to histological changes indicative of necrosis. The degree of renal apoptosis was assessed by three methods: 1) DNA fragmentation detected by terminal deoxynucleotidyl transferase biotin-dUTP nickend labeling staining, 2) DNA laddering detected after agarose gel electrophoresis, and 3) morphological identification of apoptotic tubules at the corticomedullary junction. We also measured the expression of the proinflammatory markers ICAM-1 and TNF-␣. Continuous local anesthetic infusion with renal I/R injury resulted in an increased magnitude and duration of renal dysfunction compared with the saline-infused I/R group. Additionally, both apoptotic and necrotic renal cell death as well as inflammatory changes were significantly potentiated in local anesthetic-treated rat kidneys. Local anesthetic infusion alone without I/R injury had no effect on renal function. We conclude that local anesthetics potentiated renal injury after I/R by increasing necrosis, apoptosis, and inflammation. acute renal failure; apoptosis; bupivacaine; inflammation; lidocaine; necrosis; tetracaine ACUTE RENAL FAILURE (ARF) secondary to ischemia-reperfusion (I/R) injury continues to be a significant clinical problem Patients with impaired preoperative renal function undergoing aortovascular surgery are at greatest risk for developing perioperative ARF (26). Local anesthetics are widely used in clinical practice, even in patients with impaired preoperative renal function. Epidural infusions of local anesthetic are routinely used for intraoperative and postoperative analgesia (frequently lasting several days) in patients undergoing major abdominal and vascular procedures. During induction of general anesthesia for endotracheal intubation, intravenous lidocaine is given routinely to blunt the sympathetic reflex to direct laryngoscopy. Local anesthetics are used to provide surgical anesthesia and analgesia in peripheral and central nervous system nerve blocks (spinal and epidural anesthesia). In the intensive care unit, lidocaine is frequently used as an antiarrythmic agent. Several in vitro studies found that local anesthetics increase cell death via apoptosis in neuronal, lymphocytic, and osteoblastic cell lines MATERIALS AND METHODS Implantation of Miniosmotic Pumps and Renal I/R Injury All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Adult male Sprague-Dawley rats (225-275 g, Harlan Sprague-Dawley, Indianapolis, IN) were used. They had free access to rodent chow and water. Rats were anesthetized with intraperitoneal (ip) pentobarbital sodium (50 mg/kg or to effect) and implanted with subcutaneous miniosmotic pumps (model 2ML1, Alzet) that continuously delivered 10 l/h of 5% lidocaine (2 mg⅐kg Ϫ1 ⅐h Ϫ1 ), 1% bupivacaine (0.4 mg⅐kg Ϫ1 ⅐h Ϫ1 ), 2.5% tetracaine (1 mg⅐kg Ϫ1 ⅐h Ϫ1 ), or saline vehicle. The doses of local anesthetics delivered mimicked clinically administered doses for continuous epidural infusion for a 70-kg person during and after abdominal and vascular surgical procedures. Some rats were infused with 0.5% bupivacaine instead of 1% bupivacaine. Six hours later (the time required for osmotic pump priming), rats were reanesthetized with pentobarbital sodium. After 500 U of heparin were given ip, rats were placed on an electric heating pad under a warming light. Body temperature was monitored with a rectal probe and maintained at 37°C. They were allowed to spontaneously breath room air. After a laparotomy, rats were subjected to 30-min left renal ischemia after right nephrectomy. The duration of ischemia was shown in pilot studies to produce reversible and moderate renal dysfunction in rats. Some rats were subjected to only sham operation (anesthesia, laparotomy, and right nephrectomy) and received vehicle (saline) infusion, and others received a sham operation plus local anestheti
Decay of isolated surface features driven by the Gibbs-Thomson effect in analytic model and simulation
A theory based on the thermodynamic Gibbs-Thomson relation is presented which
provides the framework for understanding the time evolution of isolated
nanoscale features (i.e., islands and pits) on surfaces. Two limiting cases are
predicted, in which either diffusion or interface transfer is the limiting
process. These cases correspond to similar regimes considered in previous works
addressing the Ostwald ripening of ensembles of features. A third possible
limiting case is noted for the special geometry of "stacked" islands. In these
limiting cases, isolated features are predicted to decay in size with a power
law scaling in time: A is proportional to (t0-t)^n, where A is the area of the
feature, t0 is the time at which the feature disappears, and n=2/3 or 1. The
constant of proportionality is related to parameters describing both the
kinetic and equilibrium properties of the surface. A continuous time Monte
Carlo simulation is used to test the application of this theory to generic
surfaces with atomic scale features. A new method is described to obtain
macroscopic kinetic parameters describing interfaces in such simulations.
Simulation and analytic theory are compared directly, using measurements of the
simulation to determine the constants of the analytic theory. Agreement between
the two is very good over a range of surface parameters, suggesting that the
analytic theory properly captures the necessary physics. It is anticipated that
the simulation will be useful in modeling complex surface geometries often seen
in experiments on physical surfaces, for which application of the analytic
model is not straightforward.Comment: RevTeX (with .bbl file), 25 pages, 7 figures from 9 Postscript files
embedded using epsf. Submitted to Phys. Rev. B A few minor changes made on
9/24/9
MicroRNA profiling of the murine hematopoietic system
BACKGROUND: MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene expression during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. RESULTS: We used oligonucleotide arrays to analyze miRNA expression in the murine hematopoietic system. Complementary oligonucleotides capable of hybridizing to 181 miRNAs were immobilized on a membrane and probed with radiolabeled RNA derived from low molecular weight fractions of total RNA from several different hematopoietic and neuronal cells. This method allowed us to analyze cell type-specific patterns of miRNA expression and to identify miRNAs that might be important for cell lineage specification and/or cell effector functions. CONCLUSION: This is the first report of systematic miRNA gene profiling in cells of the hematopoietic system. As expected, miRNA expression patterns were very different between hematopoietic and non-hematopoietic cells, with further subtle differences observed within the hematopoietic group. Interestingly, the most pronounced similarities were observed among fully differentiated effector cells (Th1 and Th2 lymphocytes and mast cells) and precursors at comparable stages of differentiation (double negative thymocytes and pro-B cells), suggesting that in addition to regulating the process of commitment to particular cellular lineages, miRNAs might have an important general role in the mechanism of cell differentiation and maintenance of cell identity
Weak selection and stability of localized distributions in Ostwald ripening
We support and generalize a weak selection rule predicted recently for the
self-similar asymptotics of the distribution function (DF) in the
zero-volume-fraction limit of Ostwald ripening (OR). An asymptotic perturbation
theory is developed that, when combined with an exact invariance property of
the system, yields the selection rule, predicts a power-law convergence towards
the selected self-similar DF and agrees well with our numerical simulations for
the interface- and diffusion-controlled OR.Comment: 4 pages, 2 figures, submitted to PR
MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors
BACKGROUND
MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated.
METHODS
Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets.
RESULTS
Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a "seedless" binding site within its 3'UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change.
CONCLUSIONS
Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers
Autoluminescent Plants
Prospects of obtaining plants glowing in the dark have captivated the imagination of scientists and layman alike. While light emission has been developed into a useful marker of gene expression, bioluminescence in plants remained dependent on externally supplied substrate. Evolutionary conservation of the prokaryotic gene expression machinery enabled expression of the six genes of the lux operon in chloroplasts yielding plants that are capable of autonomous light emission. This work demonstrates that complex metabolic pathways of prokaryotes can be reconstructed and function in plant chloroplasts and that transplastomic plants can emit light that is visible by naked eye
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