Peri-substituted hexahydro-indolones as novel,potent and selective human EP3 receptor antagonists

A series of peri-substituted [4.3.0] bicyclic non-aromatic heterocycles have been identified as potent and selective hEP₃ receptor antagonists. These molecules adopt a hair-pin conformation that overlaps with the endogenous ligand PGE₂ and fits into an internally generated EP₃ pharmacophore model. Optimized compounds show good metabolic stability and improved solubility over their corresponding bicyclic aromatic analogs.     

A Bayesian Network View on Nested Effects Models

Nested effects models (NEMs) are a class of probabilistic models that were designed to reconstruct a hidden signalling structure from a large set of observable effects caused by active interventions into the signalling pathway. We give a more flexible formulation of NEMs in the language of Bayesian networks. Our framework constitutes a natural generalization of the original NEM model, since it explicitly states the assumptions that are tacitly underlying the original version. Our approach gives rise to new learning methods for NEMs, which have been implemented in the /Bioconductor package nem. We validate these methods in a simulation study and apply them to a synthetic lethality dataset in yeast.     

Thirty-One Novel Biomarkers as Predictors for Clinically Incident Diabetes

Background

The prevalence of diabetes is increasing in all industrialized countries and its prevention has become a public health priority. However, the predictors of diabetes risk are insufficiently understood. We evaluated, whether 31 novel biomarkers could help to predict the risk of incident diabetes.

Methods and Findings

The biomarkers were evaluated primarily in the FINRISK97 cohort (n = 7,827; 417 cases of clinically incident diabetes during the follow-up). The findings were replicated in the Health 2000 cohort (n = 4,977; 179 cases of clinically incident diabetes during the follow-up). We used Cox proportional hazards models to calculate the relative risk of diabetes, after adjusting for the classic risk factors, separately for each biomarker. Next, we assessed the discriminatory ability of single biomarkers using receiver operating characteristic curves and C-statistics, integrated discrimination improvement (IDI) and net reclassification improvement (NRI). Finally, we derived a biomarker score in the FINRISK97 cohort and validated it in the Health 2000 cohort. A score consisting of adiponectin, apolipoprotein B, C-reactive protein and ferritin almost doubled the relative risk of diabetes in the validation cohort (HR per one standard deviation increase 1.88, p = 2.8 e-5). It also improved discrimination of the model (IDI = 0.0149, p<0.0001) and reclassification of diabetes risk (NRI = 11.8%, p = 0.006). Gender-specific analyses suggested that the best score differed between men and women. Among men, the best results were obtained with the score of four biomarkers: adiponectin, apolipoprotein B, ferritin and interleukin-1 receptor antagonist, which gave an NRI of 25.4% (p<0.0001). Among women, the best score included adiponectin, apolipoprotein B, C-reactive protein and insulin. It gave an NRI of 13.6% (p = 0.041).

Conclusions

We identified novel biomarkers that were associated with the risk of clinically incident diabetes over and above the classic risk factors. This gives new insights into the pathogenesis of diabetes and may help with targeting prevention and treatment.     

Effect of neighbour visibility on nest attendance patterns of Barn Swallows Hirundo rustica in loose colonies

To assess the benefits of nesting at a site hidden from neighbours in a loosely colonial species, the Barn Swallow Hirundo rustica, we carried out two field experiments, obstruction removal and mirror placement, both replicating a situation in which a nest is made visible from another nest. Under manipulated conditions in both experiments, females increased the length of time they stayed at their nests during the egg‐laying and late incubation stages, while males extended their duration of stay during the egg‐laying and early nestling stages in mirror placement experiments only. The results suggest that Barn Swallows conceal their nests to reduce fitness costs imposed by neighbours nesting in view and that hiding the nest can reduce the amount of time spent guarding the nest during certain stages of the breeding period.     

Overexpression of manganese superoxide dismutase does not increase clonogenic cell survival despite effect on apoptosis in irradiated lymphoblastoid cells

Gene therapy-mediated overexpression of superoxide dismutases (SOD) appears to be a promising strategy for modulating radiosensitivity based on detoxification of superoxide radicals and suppression of apoptosis. Using recombinant lentiviral-based vectors, the effects of SOD overexpression on both were tested in human lymphoblastoid cells (TK6) that are sensitive to radiation-induced apoptosis. TK6 cells were transduced with vectors containing CuZnSOD, MnSOD or inverted MnSOD (MSODi) cDNA. Gene transfer efficiency, SOD activity, superoxide-radical resistance, apoptosis and clonogenic survival were determined. A six- to eightfold increase in SOD activity was observed after transduction, rendering MnSOD-overexpressing TK6 cells significantly more resistant to paraquat-induced superoxide radical production than controls. Although significant differences in sensitivity to apoptosis were observed for MnSOD, no differences in clonogenic survival after irradiation were detected between any groups. Our data show that efficient cellular SOD overexpression, an increased superoxide radical detoxifying ability and, for MnSOD, decreased apoptosis did not result in increased clonogenic survival after irradiation. This strengthens the hypothesis of differences in the radiation-modulating effects of SOD on normal and malignant cells (protective and nonprotective, respectively), thereby showing its potential to increase the therapeutic index in future clinical SOD-based radioprotection approaches.     

RNA-based gene therapy for the treatment and prevention of HIV: from bench to bedside

Gene therapy is considered a feasible approach for the treatment and prevention of HIV/AIDS. Targeting both viral genes and host dependency factors can interfere with the viral lifecycle and prevent viral replication. A number of approaches have been taken to target these genes, including ribozymes, aptamers, and RNAi based therapies. A number of these therapies are now beginning to make their way into clinical trials and providing proof of principle that gene therapy is a safe and realistic option for treating HIV. Here, we focus on those therapies that have progressed along the pipeline to preclinical and clinical testing.     

Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequence-dependent regulator of translation efficiency controlling 3-phosphoinositide-dependent protein kinase-1 (PDPK-1)

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.     

Redox State of Pentraxin 3 as a Novel Biomarker for Resolution of Inflammation and Survival in Sepsis

In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung—common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the total PTX3 in non-survivors (p < 0.001). Monomeric PTX3 was inversely associated with cardiac damage markers NT-proBNP and high-sensitivity troponin I and T. Relative to the conventional measurements of total PTX3 or NT-proBNP, the oligomerization of PTX3 was a superior predictor of disease outcome.Severe sepsis is a common acute illness in intensive care units (ICUs)¹ and is associated with high mortality rates and chronic morbidity. When it is associated with hypotension (termed septic shock), the mortality rate is very high (50% to 80%). Cardiovascular dysfunction during sepsis is multifactorial and often associated with minimal loss of myocardial tissue, but with the release of myocardial-specific markers such as troponins. A key unmet clinical need is the availability of a biomarker that predicts myocardial dysfunction early, monitors response to treatment, and thus identifies a cohort of patients at higher risk of septic shock to aid in targeted interventions and improve outcome (1).In the present study, we used proteomics for biomarker discovery. Over the past decade, the field of proteomics has made impressive progress. Plasma and serum, however, are the most complex proteomes of the human body (2), and less abundant proteins tend to be missed in untargeted proteomics analyses of body fluids (3). Thus, we pursued an alternative strategy: the application of proteomics to diseased tissue (4), in which the potential biomarkers are less dilute and have a less uncertain cellular origin (5–7). We employed a solubility-based protein-subfractionation methodology to analyze inflammatory proteins that are retained with sepsis tissue. This innovative proteomics approach shall reveal inflammatory molecules that reside and persist within inflamed tissue. We hypothesized that proteins that accumulate in the susceptible tissues are more likely to be biomarker candidates for organ dysfunction than proteins that just circulate in plasma or serum. We then validated our proteomics findings in the preclinical model using samples from sepsis patients admitted to ICUs.     

Bacterial sarcosine oxidase: identification of novel substrates and a biradical reaction intermediate

Corynebacterial sarcosine oxidase contains both covalently and noncovalently bound FAD and forms complexes with various heterocyclic carboxylic acids (D-proline and 2-furoic, 2-pyrrolecarboxylic, and 2-thiophenecarboxylic acids). 2-Furoic acid, a competitive inhibitor with respect to sarcosine, selectively perturbs the absorption spectrum of the noncovalent flavin, suggesting that the enzyme has a single sarcosine binding site near the noncovalent flavin. Several heterocyclic amines have been identified as new substrates for the enzyme. Similar reactivity is observed with L-proline and L-pipecolic acid whereas L-2-azetidine-carboxylic acid is less reactive. Turnover with L-proline is slow (TN = 4.4 min-1) as compared with sarcosine (TN = 1000 min-1). Anaerobic reduction of the enzyme with heterocyclic amine substrates at pH 8.0 occurs as a biphasic reaction. A similar long-wavelength intermediate is formed in the initial fast phase of each reaction and then decays in a slower second phase to yield 1,5-dihydroFAD. The slow phase is not kinetically significant during aerobic turnover at pH 8.0 and is absent when the anaerobic reactions are conducted at pH 7.0. EPR and other studies at pH 7.0 show that the long-wavelength species is a half-reduced form of the enzyme (1 electron/substrate-reducible flavin) containing 0.9 mol of flavin radical/mol of substrate-reducible flavin. This biradical intermediate exhibits an absorption spectrum similar to that expected for a 50:50 mixture of red anionic and blue neutral flavin radicals. A similar long-wavelength species is observed during titration of the enzyme with sarcosine and other reductants. Studies with L-proline suggest that reduction of the enzyme involves initial transfer of two electrons to the noncovalent flavin. The covalent flavin is not required and can be complexed with sulfite without affecting the rate of electron transfer. The initial half-reduced form of the enzyme appears to be rapidly converted to the biradical form via comproportionation of the reduced noncovalent flavin with the oxidized covalent flavin.     

Dorso-ventral limb polarity and origin of the ridge: On the fringe of independence?

Molecular and developmental studies of limb pattern formation have recently gained widespread attention. The fact that vertebrate limbs are amenable to both genetic and embryological manipulations has established this model system as a valuable paradigm for studying vertebrate development. Limb buds are polarised along all three major axes and the establishment of the dorso-ventral (DV) polarity is dependent upon cues localised in the trunk, where a DV ectodermal interface is produced by confrontation of dorsal and ventral identities. By analogy to Drosophila imaginal disc development, this interface has been proposed to determine and position an ectodermal organising centre, the Apical Ectodermal Ridge (AER), controlling limb bud outgrowth. Recent fate mapping studies⁽¹⁾ and studies of genes regulating DV limb polarity^(2-6), AER formation^(7,8) and differentiation⁽⁹⁾ suggest, however, that DV patterning and AER induction, though coordinately regulated during limb bud outgrowth, may early on be more dissociated than expected.