Fission yeast 26S proteasome mutants are multi-drug resistant due to stabilization of the pap1 transcription factor

Here we report the result of a genetic screen for mutants resistant to the microtubule poison methyl benzimidazol-2-yl carbamate (MBC) that were also temperature sensitive for growth. In total the isolated mutants were distributed in ten complementation groups. Cloning experiments revealed that most of the mutants were in essential genes encoding various 26S proteasome subunits. We found that the proteasome mutants are multi-drug resistant due to stabilization of the stress-activated transcription factor Pap1. We show that the ubiquitylation and ultimately the degradation of Pap1 depend on the Rhp6/Ubc2 E2 ubiquitin conjugating enzyme and the Ubr1 E3 ubiquitin-protein ligase. Accordingly, mutants lacking Rhp6 or Ubr1 display drug-resistant phenotypes

Limits on scalar leptoquark interactions and consequences for GUTs

A colored weak singlet scalar state with hypercharge 4/3 is one of the possible candidates for the explanation of the unexpectedly large forward-backward asymmetry in t tbar production as measured by the CDF and D0 experiments. We investigate the role of this state in a plethora of flavor changing neutral current processes and precision observables of down-quarks and charged leptons. Our analysis includes tree- and loop-level mediated observables in the K and B systems, the charged lepton sector, as well as the Z to b bbar decay width. We perform a global fit of the relevant scalar couplings. This approach can explain the (g-2)_mu anomaly while tensions among the CP violating observables in the quark sector, most notably the nonstandard CP phase (and width difference) in the Bs system cannot be fully relaxed. The results are interpreted in a class of grand unified models which allow for a light colored scalar with a mass below 1TeV. We find that the renormalizable SU(5) scenario is not compatible with our global fit, while in the SO(10) case the viability requires the presence of both the 126- and 120-dimensional representations.Comment: 26 pages, 7 figures; version as publishe

Totally biological composite aortic stentless valved conduit for aortic root replacement: 10-year experience

<p>Abstract</p> <p>Objectives</p> <p>To retrospectively analyze the clinical outcome of a totally biological composite stentless aortic valved conduit (No-React^®BioConduit) implanted using the Bentall procedure over ten years in a single centre.</p> <p>Methods</p> <p>Between 27/10/99 and 19/01/08, the No-React^®BioConduit composite graft was implanted in 67 patients. Data on these patients were collected from the in-hospital database, from patient notes and from questionnaires. A cohort of patients had 2D-echocardiogram with an average of 4.3 ± 0.45 years post-operatively to evaluate valve function, calcification, and the diameter of the conduit.</p> <p>Results</p> <p>Implantation in 67 patients represented a follow-up of 371.3 patient-year. Males were 60% of the operated population, with a mean age of 67.9 ± 1.3 years (range 34.1-83.8 years), 21 of them below the age of 65. After a mean follow-up of 7.1 ± 0.3 years (range of 2.2-10.5 years), more than 50% of the survivors were in NYHA I/II and more than 60% of the survivors were angina-free (CCS 0). The overall 10-year survival following replacement of the aortic valve and root was 51%. During this period, 88% of patients were free from valved-conduit related complications leading to mortality. Post-operative echocardiography studies showed no evidence of stenosis, dilatation, calcification or thrombosis. Importantly, during the 10-year follow-up period no failures of the valved conduit were reported, suggesting that the tissue of the conduit does not structurally change (histology of one explant showed normal cusp and conduit).</p> <p>Conclusions</p> <p>The No-React^®BioConduit composite stentless aortic valved conduit provides excellent long-term clinical results for aortic root replacement with few prosthesis-related complications in the first post-operative decade.</p

Early prediction of treatment response to high-dose salvage chemotherapy in patients with relapsed germ cell cancer using [18F]FDG PET

To assess the ability of [18F]fluorodeoxyglucose positron emission tomography for the early prediction of response in patients with relapsed metastatic germ cell tumours undergoing salvage high-dose chemotherapy. The role of positron emission tomography was compared with established means of tumour response assessment such as CT scans/MRI and serum tumour marker changes. In addition, positron emission tomography was compared with a current prognostic score which differentiates three prognostic groups with failure-free survival rates ranging from 5–50%. [18F]fluorodeoxyglucose uptake of metastases from germ cell tumours as well as CT scans and serum tumour marker were acquired after 2–3 cycles of induction chemotherapy but before the start of high-dose chemotherapy and CT scans/serum tumour marker were compared with the baseline examinations in 23 patients with relapsed germ cell tumours. To evaluate the validity of early response prediction by positron emission tomography, radiological monitoring and serum tumour marker decline, histopathologic response after resection of residual masses and/or the clinical course over 6 months after the end of treatment (relapse vs freedom of progression) were used. Overall, 10 patients (43%) achieved a marker-negative partial remission, three (13%) a marker-positive partial remission, five (22%) a disease stabilization and five (22%) progressed during treatment. Nine patients (39%) remained progression-free over 6 months following treatment, whereas 14 (61%) progressed. The outcome of high-dose chemotherapy was correctly predicted by positron emission tomography/CT scan/serum tumour marker in 91/59/48%. Eight patients with a favourably predicted outcome by CT scans plus serum tumour marker but a positive positron emission tomography prior to high-dose chemotherapy, failed treatment. This results in the following sensitivities/specificities for the prediction of failure of high-dose chemotherapy: positron emission tomography 100/78%; radiological monitoring 43/78%; serum tumour marker 15/100%. The positive and negative predictive values of positron emission tomography were 88 and 100%, respectively. As compared with the prognostic score, positron emission tomography was correctly positive in all patients of the three risk groups who failed treatment. In addition, a negative positron emission tomography correctly predicted a favourable outcome in the good and intermediate group. [18F]fluorodeoxyglucose positron emission tomography imaging can be used to assess response to chemotherapy in patients with relapsed germ cell tumours early in the course of treatment and may help to identify patients most likely to achieve a favourable response to subsequent high-dose chemotherapy. In patients with response to induction chemotherapy according to CT scans or serum tumour marker evaluation, positron emission tomography seems to add information to detect patients with an overall unfavourable outcome. It may also be a valuable addition to the prognostic model particularly in the good and intermediate group for further selection of patients who will profit from high-dose chemotherapy

Rational Design of Temperature-Sensitive Alleles Using Computational Structure Prediction

Temperature-sensitive (ts) mutations are mutations that exhibit a mutant phenotype at high or low temperatures and a wild-type phenotype at normal temperature. Temperature-sensitive mutants are valuable tools for geneticists, particularly in the study of essential genes. However, finding ts mutations typically relies on generating and screening many thousands of mutations, which is an expensive and labor-intensive process. Here we describe an in silico method that uses Rosetta and machine learning techniques to predict a highly accurate “top 5” list of ts mutations given the structure of a protein of interest. Rosetta is a protein structure prediction and design code, used here to model and score how proteins accommodate point mutations with side-chain and backbone movements. We show that integrating Rosetta relax-derived features with sequence-based features results in accurate temperature-sensitive mutation predictions

Convergence of Rad6/Rad18 and Fanconi Anemia Tumor Suppressor Pathways upon DNA Damage

Extremely high cancer incidence associated with patients with Fanconi anemia (FA) suggests the importance of the FA signaling pathway in the suppression of non-FA human tumor development. Indeed, we found that an impaired FA signaling pathway substantially contributes to the development of non-FA human tumors. However, the mechanisms underlying the function of the FA pathway remain less understood. Using RNA interfering approach in combining with cell proliferation and reporter assays, we showed that the function of FA signaling pathway is at least partly mediated through coupling with hRad6/hRad18 signaling (HHR6 pathway). We previously reported that FANCD2 monoubiquitination, a hallmark of the FA pathway activation, can be regulated by HHR6. Here we found that hRad18 can also regulate activation of the FA pathway. More importantly, we found that FANCD2 is capable of modulating activity of DNA translesion synthesis polymerase eta, an effector of HHR6 pathway. These results provide novel insights into how the FA pathway is intertwined with HHR6 pathway to maintain chromosomal stability and suppress the development of human cancer, representing an important conceptual advance in the field of FA cancer research

Delegation and coordination with multiple threshold public goods: experimental evidence

When multiple charities, social programs and community projects simultaneously vie for funding, donors risk mis-coordinating their contributions leading to an inefficient distribution of funding across projects. Community chests and other intermediary organizations facilitate coordination among donors and reduce such risks. To study this, we extend a threshold public goods framework to allow donors to contribute through an intermediary rather than directly to the public goods. Through a series of experiments, we show that the presence of an intermediary increases public good success and subjects’ earnings only when the intermediary is formally committed to direct donations to socially beneficial goods. Without such a restriction, the presence of an intermediary has a negative impact, complicating the donation environment, decreasing contributions and public good success.When multiple charities, social programs and community projects simultaneously vie for funding, donors risk mis-coordinating their contributions leading to an inefficient distribution of funding across projects. Community chests and other intermediary organizations facilitate coordination among donors and reduce such risks. To study this, we extend a threshold public goods framework to allow donors to contribute through an intermediary rather than directly to the public goods. Through a series of experiments, we show that the presence of an intermediary increases public good success and subjects’ earnings only when the intermediary is formally committed to direct donations to socially beneficial goods. Without such a restriction, the presence of an intermediary has a negative impact, complicating the donation environment, decreasing contributions and public good success

Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of Monocyte Migration in Immune-Mediated Inflammatory Disease

Background: Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man. Methods/Principal Findings: We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (99m-Tc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4x10(-3) (0.95-5.1x10(-3)) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion. Conclusions/Significance: The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention

Dibucaine Mitigates Spreading Depolarization in Human Neocortical Slices and Prevents Acute Dendritic Injury in the Ischemic Rodent Neocortex

Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury.We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model.Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations

The Polycomb Repressive Complex 2 Is a Potential Target of SUMO Modifications

The Polycomb Repressive Complex 2 (PRC2) functions as a transcriptional repressor through a mechanism that involves methylation of Histone H3 at lysine 27. The PRC2 complex activity is essential for cellular proliferation, development, and cell fate decisions. PRC2 target genes include important regulators of development and proliferation as well as tumor suppressor genes. Consistent with this, the activity of several Polycomb group (PcG) proteins is deregulated in human cancer suggesting an important role for PcGs in tumor development. Whereas the downstream functions of PcGs are well characterized, the mechanisms of their recruitment to target genes and the regulation of their activity are not fully understood.Here we show that the two PRC2 components SUZ12 and EZH2 are sumoylated in vitro and in vivo. Among several putative sumoylation sites we have mapped the major site of SUZ12 sumoylation. Furthermore, we show that SUZ12 interacts with the E2-conjugating enzyme UBC9 both in vitro and in vivo and that mutation of the SUZ12 sumoylation site does not abolish this binding. Finally, we provide evidence that the E3-ligase PIASXbeta interacts and enhances the sumoylation of SUZ12 in vivo suggesting that PIASXbeta could function as an E3-ligase for SUZ12.Taken together, our data identify sumoylation as a novel post-translational modification of components of the PRC2 complex, which could suggest a potential new mechanism to modulate PRC2 repressive activity. Further work aimed to identify the physiological conditions for these modifications will be required to understand the role of SUZ12 and EZH2 sumoylation in PcG-mediated epigenetic regulation of transcription