Evidence for Two Modes of Synergistic Induction of Apoptosis by Mapatumumab and Oxaliplatin in Combination with Hyperthermia in Human Colon Cancer Cells

Colorectal cancer is the third leading cause of cancer-related mortality in the world-- the main cause of death from colorectal cancer is hepatic metastases, which can be treated with isolated hepatic perfusion (IHP). Searching for the most clinically relevant approaches for treating colorectal metastatic disease by isolated hepatic perfusion (IHP), we developed the application of oxaliplatin concomitantly with hyperthermia and humanized death receptor 4 (DR4) antibody mapatumumab (Mapa), and investigated the molecular mechanisms of this multimodality treatment in human colon cancer cell lines CX-1 and HCT116 as well as human colon cancer stem cells Tu-12, Tu-21 and Tu-22. We showed here, in this study, that the synergistic effect of the multimodality treatment-induced apoptosis was caspase dependent and activated death signaling via both the extrinsic apoptotic pathway and the intrinsic pathway. Death signaling was activated by c-Jun N-terminal kinase (JNK) signaling which led to Bcl-xL phosphorylation at serine 62, decreasing the anti-apoptotic activity of Bcl-xL, which contributed to the intrinsic pathway. The downregulation of cellular FLICE inhibitory protein long isoform (c-FLIPL) in the extrinsic pathway was accomplished through ubiquitination at lysine residue (K) 195 and protein synthesis inhibition. Overexpression of c-FLIPL mutant (K195R) and Bcl-xL mutant (S62A) completely abrogated the synergistic effect. The successful outcome of this study supports the application of multimodality strategy to patients with colorectal hepatic metastases who fail to respond to standard chemoradiotherapy that predominantly targets the mitochondrial apoptotic pathway. © 2013 Song et al

Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

<p>Abstract</p> <p>Background</p> <p>Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine.</p> <p>Methods</p> <p>GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition.</p> <p>Results</p> <p>GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-X_L, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic.</p> <p>Conclusion</p> <p>GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be context or cell line dependent, but could also be explained on the basis that although NF-kappaB is an important mediator of pancreatic cancer cell survival, it plays a minor role in gemcitabine resistance. Further work is needed to understand the mechanisms of this effect, including the potential for rational combination of GSK3 inhibitors with other targeted agents for the treatment of pancreatic cancer.</p

Reduction of the ATPase inhibitory factor 1 (IF1) leads to visual impairment in vertebrates

In vertebrates, mitochondria are tightly preserved energy producing organelles, which sustain nervous system development and function. The understanding of proteins that regulate their homoeostasis in complex animals is therefore critical and doing so via means of systemic analysis pivotal to inform pathophysiological conditions associated with mitochondrial deficiency. With the goal to decipher the role of the ATPase inhibitory factor 1 (IF1) in brain development, we employed the zebrafish as elected model reporting that the Atpif1a−/− zebrafish mutant, pinotage (pnttq209), which lacks one of the two IF1 paralogous, exhibits visual impairment alongside increased apoptotic bodies and neuroinflammation in both brain and retina. This associates with increased processing of the dynamin-like GTPase optic atrophy 1 (OPA1), whose ablation is a direct cause of inherited optic atrophy. Defects in vision associated with the processing of OPA1 are specular in Atpif1−/− mice thus confirming a regulatory axis, which interlinks IF1 and OPA1 in the definition of mitochondrial fitness and specialised brain functions. This study unveils a functional relay between IF1 and OPA1 in central nervous system besides representing an example of how the zebrafish model could be harnessed to infer the activity of mitochondrial proteins during development

Evidence for the h_b(1P) meson in the decay Upsilon(3S) --> pi0 h_b(1P)

Using a sample of 122 million Upsilon(3S) events recorded with the BaBar detector at the PEP-II asymmetric-energy e+e- collider at SLAC, we search for the $h_b(1P)$ spin-singlet partner of the P-wave chi_{bJ}(1P) states in the sequential decay Upsilon(3S) --> pi0 h_b(1P), h_b(1P) --> gamma eta_b(1S). We observe an excess of events above background in the distribution of the recoil mass against the pi0 at mass 9902 +/- 4(stat.) +/- 2(syst.) MeV/c^2. The width of the observed signal is consistent with experimental resolution, and its significance is 3.1sigma, including systematic uncertainties. We obtain the value (4.3 +/- 1.1(stat.) +/- 0.9(syst.)) x 10^{-4} for the product branching fraction BF(Upsilon(3S)-->pi0 h_b) x BF(h_b-->gamma eta_b).Comment: 8 pages, 4 postscript figures, submitted to Phys. Rev. D (Rapid Communications

Assessment of Medical Students’ Shared Decision-Making in Standardized Patient Encounters

BackgroundShared decision-making, in which physicians and patients openly explore beliefs, exchange information, and reach explicit closure, may represent optimal physician-patient communication. There are currently no universally accepted methods to assess medical students' competence in shared decision-making.ObjectiveTo characterize medical students' shared decision-making with standardized patients (SPs) and determine if students' use of shared decision-making correlates with SP ratings of their communication.DesignRetrospective study of medical students' performance with four SPs.ParticipantsSixty fourth-year medical students.MeasurementsObjective blinded coding of shared decision-making quantified as decision moments (exploration/articulation of perspective, information sharing, explicit closure for a particular decision); SP scoring of communication skills using a validated checklist.ResultsOf 779 decision moments generated in 240 encounters, 312 (40%) met criteria for shared decision-making. All students engaged in shared decision-making in at least two of the four cases, although in two cases 5% and 12% of students engaged in no shared decision-making. The most commonly discussed decision moment topics were medications (n = 98, 31%), follow-up visits (71, 23%), and diagnostic testing (44, 14%). Correlations between the number of decision moments in a case and students' communication scores were low (rho = 0.07 to 0.37).ConclusionsAlthough all students engaged in some shared decision-making, particularly regarding medical interventions, there was no correlation between shared decision-making and overall communication competence rated by the SPs. These findings suggest that SP ratings of students' communication skill cannot be used to infer students' use of shared decision-making. Tools to determine students' skill in shared decision-making are needed

An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available

Zebrafish Numb and Numblike Are Involved in Primitive Erythrocyte Differentiation

BACKGROUND:Notch signaling is an evolutionarily conserved regulatory circuitry implicated in cell fate determination in various developmental processes including hematopoietic stem cell self-renewal and differentiation of blood lineages. Known endogenous inhibitors of Notch activity are Numb-Nb and Numblike-Nbl, which play partially redundant functions in specifying and maintaining neuronal differentiation. Nb and Nbl are expressed in most tissues including embryonic and adult hematopoietic tissues in mice and humans, suggesting possible roles for these proteins in hematopoiesis. METHODOLOGY AND PRINCIPAL FINDINGS:We employed zebrafish to investigate the possible functional role of Numb and Numblike during hematopoiesis, as this system allows a detailed analysis even in embryos with severe defects that would be lethal in other organisms. Here we describe that nb/nbl knockdown results in severe reduction or absence of embryonic erythrocytes in zebrafish. Interestingly, nb/nbl knocked-down embryos present severe downregulation of the erythroid transcription factor gata1. This results in erythroblasts which fail to mature and undergo apoptosis. Our results indicate that Notch activity is increased in embryos injected with nb/nbl morpholino, and we show that inhibition of Notch activation can partially rescue the hematopoietic phenotype. CONCLUSIONS AND SIGNIFICANCE:Our results provide the first in vivo evidence of an involvement of Numb and Numblike in zebrafish erythroid differentiation during primitive hematopoiesis. Furthermore, we found that, at least in part, the nb/nbl morphant phenotype is due to enhanced Notch activation within hematopoietic districts, which in turn results in primitive erythroid differentiation defects

The Mechanism for RNA Recognition by ANTAR Regulators of Gene Expression

ANTAR proteins are widespread bacterial regulatory proteins that have RNA–binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR–associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism

Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

BACKGROUND: Recent studies have suggested that periodontal disease increases the risk of atherothrombotic disease. Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in the arteries. Although several studies have suggested that certain periodontopathic bacteria accelerate atherogenesis in apolipoprotein E-deficient mice, the mechanistic link between cholesterol accumulation and periodontal infection-induced inflammation is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We orally infected C57BL/6 and C57BL/6.KOR-Apoe(shl) (B6.Apoeshl) mice with Porphyromonas gingivalis, which is a representative periodontopathic bacterium, and evaluated atherogenesis, gene expression in the aorta and liver and systemic inflammatory and lipid profiles in the blood. Furthermore, the effect of lipopolysaccharide (LPS) from P. gingivalis on cholesterol transport and the related gene expression was examined in peritoneal macrophages. Alveolar bone resorption and elevation of systemic inflammatory responses were induced in both strains. Despite early changes in the expression of key genes involved in cholesterol turnover, such as liver X receptor and ATP-binding cassette A1, serum lipid profiles did not change with short-term infection. Long-term infection was associated with a reduction in serum high-density lipoprotein (HDL) cholesterol but not with the development of atherosclerotic lesions in wild-type mice. In B6.Apoeshl mice, long-term infection resulted in the elevation of very low-density lipoprotein (VLDL), LDL and total cholesterols in addition to the reduction of HDL cholesterol. This shift in the lipid profile was concomitant with a significant increase in atherosclerotic lesions. Stimulation with P. gingivalis LPS induced the change of cholesterol transport via targeting the expression of LDL receptor-related genes and resulted in the disturbance of regulatory mechanisms of the cholesterol level in macrophages. CONCLUSIONS/SIGNIFICANCE: Periodontal infection itself does not cause atherosclerosis, but it accelerates it by inducing systemic inflammation and deteriorating lipid metabolism, particularly when underlying hyperlidemia or susceptibility to hyperlipidemia exists, and it may contribute to the development of coronary heart disease

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012