Search

The public administration represents the subsector of one of the 12 sectors with critical infrastructures in Romania, namely the Administration sector. Its role is to ensure the organization and concrete execution of the law, in order to protect the rights and freedoms of citizens. The efficiency and performance of the public administration is mainly determined by the effectiveness of the civil servant. That is why the human factor is the main resource in the administration sector, being also the most vulnerable element of this sector. The unpredictability of human resources makes measures to reduce the vulnerabilities caused by it (human resources) the most difficult to manage.

The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.

UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13L’s C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in unc-10 mutants deficient for C2A/RIM heterodimerization, leading to decreased release probability. Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.

UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13L’s C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in unc-10 mutants deficient for C2A/RIM heterodimerization, leading to decreased release probability. Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.

Summary The synaptic adhesion molecules Neurexin and Neuroligin alter the development and function of synapses and are linked to autism in humans. In C. elegans , post-synaptic Neurexin (NRX-1) and pre-synaptic Neuroligin (NLG-1) mediate a retrograde synaptic signal that inhibits acetylcholine (ACh) release at neuromuscular junctions. Here, we show that the retrograde signal decreases ACh release by inhibiting the function of pre-synaptic UNC-2/CaV2 calcium channels. Post-synaptic NRX-1 binds to an auxiliary subunit of pre-synaptic UNC-2/CaV2 channels (UNC-36/α2δ), decreasing UNC-36 abundance at pre-synaptic elements. Retrograde inhibition is mediated by a soluble form of NRX-1's ectodomain, which is released from the post-synaptic membrane by the SUP-17/ADAM10 protease. Mammalian Neurexin-1α binds α2δ-3 and decreases CaV2.2 current in transfected cells, whereas Neurexin-1α has no effect on CaV2.2 reconstituted with α2δ-1 and α2δ-2. Collectively, these results suggest that α-Neurexin binding to α2δ is a conserved mechanism for regulating synaptic transmission.

Melanoma tumors usually retain wild-type p53; however, its tumor-suppressor activity is functionally disabled, most commonly through an inactivating interaction with mouse double-minute 2 homolog (Mdm2), indicating p53 release from this complex as a potential therapeutic approach. P53 and the tumor-promoter insulin-like growth factor type 1 receptor (IGF-1R) compete as substrates for the E3 ubiquitin ligase Mdm2, making their relative abundance intricately linked. Hence we investigated the effects of pharmacological Mdm2 release from the Mdm2/p53 complex on the expression and function of the IGF-1R. Nutlin-3 treatment increased IGF-1R/Mdm2 association with enhanced IGF-1R ubiquitination and a dual functional outcome: Receptor downregulation and selective downstream signaling activation confined to the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. This Nutlin-3 functional selectivity translated into IGF-1-mediated bioactivities with biphasic effects on the proliferative and metastatic phenotype: An early increase and late decrease in the number of proliferative and migratory cells, while the invasiveness was completely inhibited following Nutlin-3 treatment through an impaired IGF-1-mediated matrix metalloproteinases type 2 activation mechanism. Taken together, these experiments reveal the biased agonistic properties of Nutlin-3 for the mitogen-activated protein kinase pathway, mediated by Mdm2 through IGF-1R ubiquitination and provide fundamental insights into destabilizing p53/Mdm2/IGF-1R circuitry that could be developed for therapeutic gain.

Purpose: To assess the value of contrast-enhanced ultrasound (CEUS) for differentiating malignant from benign focal liver lesions (FLLs) and for diagnosing different FLL types. Material and Methods: CEUS performed in 14 Romanian centers was prospectively collected between February 2011 and June 2012. The inclusion criteria were: age > 18 years; patients diagnosed with 1 – 3 de novo FLLs on B-mode ultrasound; reference method (computed tomography (CT), magnetic resonance imaging (MRI) or biopsy) available; patient’s informed consent. FLL lesions were characterized during CEUS according to the European Federation of Societies for Ultrasound in Medicine and Biology guidelines. For statistical analysis, indeterminate FLLs at CEUS were rated as false classifications. Results: A total number of 536 cases were included in the final analysis, 344 malignant lesions (64.2 %) and 192 benign lesions (35.8 %). The reference method was: CT/MRI – 379 cases (70.7 %), pathological exam – 150 cases (27.9 %) and aspiration of liver abscesses – 7 cases (1.4 %). CEUS was conclusive in 89.3 % and inconclusive in 10.7 % of cases. To differentiate between malignant and benign FLLs, CEUS had 85.7 % sensitivity, 85.9 % specificity, 91.6 % positive predictive value, 77.1 % negative predictive value and 85.8 % accuracy. The CEUS accuracy for differentiation between malignant and benign liver lesions was similar in tumors with diameter ≤ 2 cm and those with diameter > 2 cm. Conclusion: CEUS represents a useful method in clinical practice for differentiating between malignant and benign FLLs detected on standard ultrasonography, and the results of this study are in concordance with previous multicenter studies: DEGUM (Germany) and STIC (France).

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.

Oxysterols bind the seven-spanner transmembrane protein Smoothened and potently activate vertebrate Hedgehog signaling, a pathway essential in embryonic development, adult stem cell maintenance and cancer. It is unknown, however, if oxysterols are important for normal vertebrate Hedgehog signaling, and whether antagonizing oxysterols can inhibit the Hedgehog pathway. We developed azasterols that block Hedgehog signaling by binding the oxysterol-binding site of Smoothened. We show that the binding site for oxysterols and azasterols maps to the extracellular, cysteine-rich domain of Smoothened, and is completely separable from the site bound by other small molecule modulators, located within the heptahelical bundle of Smoothened. Smoothened mutants in which oxysterol binding is abolished no longer respond to oxysterols, and cannot be maximally activated by the Hedgehog ligand. Our results show that oxysterol binding to vertebrate Smoothened is required for normal Hedgehog signaling, and that targeting the oxysterol binding site is an effective strategy to inhibit Smoothened.

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo . Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.