23 research outputs found
Identifying Drug Effects via Pathway Alterations using an Integer Linear Programming Optimization Formulation on Phosphoproteomic Data
Understanding the mechanisms of cell function and drug action is a major endeavor in
the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the
drug (i.e., selectivity and potency) and the specific signaling transduction network of the
host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomicbased
approach to identify drug effects by monitoring drug-induced topology alterations.
With the proposed method, drug effects are investigated under several conditions on a
cell-type specific signaling network. First, starting with a generic pathway made of
logical gates, we build a cell-type specific map by constraining it to fit 13 key
phopshoprotein signals under 55 experimental cases. Fitting is performed via a
formulation as an Integer Linear Program (ILP) and solution by standard ILP solvers; a
procedure that drastically outperforms previous fitting schemes. Then, knowing the cell
topology, we monitor the same key phopshoprotein signals under the presence of drug
and cytokines and we re-optimize the specific map to reveal the drug-induced topology
alterations. To prove our case, we make a pathway map for the hepatocytic cell line
HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal
Growth Factor Receptor (EGFR) and a non selective drug. We confirm effects easily
predictable from the drugs’ main target (i.e. EGFR inhibitors blocks the EGFR pathway)
but we also uncover unanticipated effects due to either drug promiscuity or the cell’s
specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib is
able to inhibit signaling downstream the Interleukin-1alpha (IL-1α) pathway; an effect
that cannot be extracted from binding affinity based approaches. Our method represents
an unbiased approach to identify drug effects on a small to medium size pathways and
is scalable to larger topologies with any type of signaling perturbations (small molecules,
3
RNAi etc). The method is a step towards a better picture of drug effects in pathways,
the cornerstone in identifying the mechanisms of drug efficacy and toxicity
Transesophageal echocardiography for diagnosis of inadvertant closure of Inferior Vena Cava opening during minimally invasive atrial septal defect closure
Transesophageal echocardiography (TEE) is widely used in cardiac surgery. TEE provides important diagnostic and functional information before and after cardiopulmonary bypass thereby having a very important impact on perioperative clinical outcomes. We describe a case in which intraoperative TEE was instrumental in the timely diagnosis of inadvertant closure of the inferior vena cava (IVC) opening during minimally invasive surgical closure of atrial septal defect
Controlling costs in the intensive-care unit: role of daily microbiologist rounds in an Indian hospital
A Methodology for Assessing the Probability of Occurrence of Undesired Events in the Tietê–Paraná Inland Waterway Based on Expert Opinion
Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo
Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T -> Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S -> A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (similar to 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h. FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h. FIX: Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B