Combination of a Proteomics Approach and Reengineering of Meso Scale Network Models for Prediction of Mode-of-Action for Tyrosine Kinase Inhibitors

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.     

The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics

Cyclic AMP (cAMP)-dependent phosphorylation has been reported to exert biological effects in both the mitochondrial matrix and outer mitochondrial membrane (OMM). However, the kinetics, targets, and effectors of the cAMP cascade in these organellar domains remain largely undefined. Here we used sensitive FRET-based sensors to monitor cAMP and protein kinase A (PKA) activity in different mitochondrial compartments in real time. We found that cytosolic cAMP did not enter the matrix, except during mitochondrial permeability transition. Bicarbonate treatment (expected to activate matrix-bound soluble adenylyl cyclase) increased intramitochondrial cAMP, but along with membrane-permeant cAMP analogues, failed to induce measureable matrix PKA activity. In contrast, the OMM proved to be a domain of exceptionally persistent cAMP-dependent PKA activity. Although cAMP signaling events measured on the OMM mirrored those of the cytosol, PKA phosphorylation at the OMM endured longer as a consequence of diminished control by local phosphatases. Our findings demonstrate that mitochondria host segregated cAMP cascades with distinct functional and kinetic signatures.     

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.     

Extrakorporale Membranoxygenierung in der Intensivmedizin

Extracorporeal membrane oxygenation (ECMO) represents a valuable tool in the armamentarium of life and organ support measures in intensive care medicine. ECMO is used in fulminant pulmonary failure to prevent acute hypoxia and to allow CO₂ removal (veno-venous ECMO) or, in highly selected cases of acute cardiovascular failure, to maintain organ perfusion and gas exchange and to prevent imminent death (veno-arterial ECMO). The purpose of ECMO is to allow time for intrinsic recovery of the lungs and heart. Alternatively, ECMO can be used as bridging device until a suitable organ for transplantation is available or the implantation of an artificial heart is feasible. ECMO carries a high complication rate and its use should be limited to highly specialized centers. In rare cases, mobile ECMO systems can be used by dedicated teams which allow transportation of critically ill patients over longer distances to specialized institutions. There is no unequivocal evidence from controlled studies that the use of ECMO in adults improves survival in larger cohorts, but its use in selected cases can be life saving. Long-term survivors of ECMO therapy report significant impairments in health-related quality of life (HRQL) when compared to healthy controls. There is, however, a gradual recovery and improvement in HRQL over a prolonged period after discharge from the intensive care unit and severe limitations in cognitive or physical function are rare. The incidence of chronic post-traumatic stress disorder (PTSD) in patients after ECMO can reach up to 30%. PTSD has a major negative impact on HRQL outcomes of ECMO therapy. PTSD or other stress-related disorders need to be diagnosed and adequately treated to allow adequate recovery from the extreme illness these patients have survived.     

Clinical Profiles and Factors Associated with Death in Adults with Dengue Admitted to Intensive Care Units,Minas Gerais,Brazil

The purpose of our study was to describe the clinical profile of dengue-infected patients admitted to Brazilian intensive care units (ICU) and evaluate factors associated with death. A longitudinal, multicenter case series study was conducted with laboratory-confirmed dengue patients admitted to nine Brazilian ICUs situated in Minas Gerais state, southeastern Brazil from January 1, 2008, to December 31, 2013. Demographic, clinical and laboratory data; disease severity scores; and mortality were evaluated. A total of 97 patients were studied. The in-ICU and in-hospital mortality rates were 18.6% and 19.6%, respectively. Patients classified as having severe dengue according to current World Health Organization classifications showed an increased risk of death in a univariate analysis. Nonsurvivors were older, exhibited lower serum albumin concentrations and higher total leukocyte counts and serum creatinine levels. Other risk factors (vomiting, lethargy/restlessness, dyspnea/respiratory distress) were also associated with death in a univariate analysis. Multivariate analysis indicated that in-hospital mortality was significantly associated with Acute Physiology and Chronic Health Evaluation II and the Sequential Organ Failure Assessment score. The ICU and in-hospital mortality observed in this study were higher than values reported in similar studies. An increased frequency of ICU admission due to severe organ dysfunction, higher severity indices and scarcity of ICU beds may partially explain the higher mortality.     

Synthesis and biological evaluation of new active For‐Met‐Leu‐Phe‐OMe analogues containing para‐substituted Phe residues

In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH₃ and ‐C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics.     

HIV and STI Prevalence among Female Sex Workers in C?te d'Ivoire: Why Targeted Prevention Programs Should Be Continued and Strengthened

Objective

To assess condom use and prevalence of STIs and HIV among female sex workers (FSWs), as part of a comprehensive monitoring and evaluation plan of a nationwide sex worker prevention project in Côte d''Ivoire.

Design and Methods

Cross sectional surveys were conducted among FSWs attending five project clinics in Abidjan and San Pedro (2007), and in Yamoussoukro and Gagnoa (2009). A standardized questionnaire was administered in a face-to-face interview, which included questions on socio-demographic characteristics, sexual behaviour and condom use. After the interview, the participants were asked to provide samples for STI and HIV testing.

Results

A total of 1110 FSWs participated in the surveys. There were large differences in socio-demographic and behavioural characteristics between FSW coming for the first time as compared to FSW coming on a routine visit. The prevalence of N. gonorrhoeae or C.trachomatis was 9.1%, 11.8% among first vs. 6.9% routine attendees (p = 0.004). The overall HIV prevalence was 26.6%, it was lower among first time attendees (17.5% as compared to 33.9% for routine attendees, p<0.001). The HIV prevalence among first attendees was also lower than the proportion of HIV positive tests from routine testing and counselling services in the same clinics.

Conclusions

The results show a relatively high STI and HIV prevalence among FSWs in different cities in Côte d''Ivoire. In the light of these results, prevention efforts should continue to focus on FSWs in the country.