Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) drives viral B cell transformation and oncogenesis. LMP1's transforming activity depends on its C-terminal activation region 2 (CTAR2), which induces NF-κB and JNK by engaging TNF receptor-associated factor 6 (TRAF6). The mechanism of TRAF6 recruitment to LMP1 and its role in LMP1 signalling remains elusive. Here we demonstrate that TRAF6 interacts directly with a viral TRAF6 binding motif within CTAR2. Functional and NMR studies supported by molecular modeling provide insight into the architecture of the LMP1-TRAF6 complex, which differs from that of CD40-TRAF6. The direct recruitment of TRAF6 to LMP1 is essential for NF-κB activation by CTAR2 and the survival of LMP1-driven lymphoma. Disruption of the LMP1-TRAF6 complex by inhibitory peptides interferes with the survival of EBV-transformed B cells. In this work, we identify LMP1-TRAF6 as a critical virus-host interface and validate this interaction as a potential therapeutic target in EBV-associated cancer.

Water envelope has a critical impact on the design of protein-protein interaction inhibitors.

We show that a water envelope network plays a critical role in protein-protein interactions (PPI). The potency of a PPI inhibitor is modulated by orders of magnitude on manipulation of the solvent envelope alone. The structure-activity relationship of PEX14 inhibitors was analyzed as an example using in silico and X-ray data.

Structure-Activity Relationship in Pyrazolo[4,3-c]pyridines, First Inhibitors of PEX14-PEX5 Protein-Protein Interaction with Trypanocidal Activity.

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against Trypanosoma, including the human pathogen Trypanosoma brucei gambiense and Trypanosoma cruzi parasites.

Structural Insights into BET Client Recognition of Endometrial and Prostate Cancer-Associated SPOP Mutants.

BET proteins such as BRD3 are oncogenic transcriptional coactivators. SPOP binding triggers their proteasomal degradation. In both endometrial and prostate cancers, SPOP mutations occur in the MATH domain, but with opposed influence on drug susceptibility. In prostate cancer, SPOP mutations presumably cause increased BET levels, decreasing BET inhibitor drug susceptibility. As opposed, in endometrial cancer, decreased BET levels concomitant with higher BET inhibitor drug susceptibility were observed. Here, we present the to our knowledge first co-crystal structure of SPOP and a bromodomain containing protein (BRD3). Our structural and biophysical data confirm the suggested loss-of-function in prostate cancer-associated SPOP mutants and provide mechanistic explanation. As opposed to previous literature, our data on endometrial cancer-associated SPOP mutants do not show altered binding behavior compared to wild-type SPOP, indicating a more complex regulatory mechanism. SPOP mutation screening may thus be considered a valuable personalized medicine tool for effective antitumor therapy.

The Structure of the SPOP-Pdx1 Interface Reveals Insights into the Phosphorylation-Dependent Binding Regulation.

Pdx1 is a transcription factor crucial for development and maintenance of a functional pancreas. It regulates insulin expression and glucose homeostasis. SPOP is an E3-ubiquitin ligase adaptor protein that binds Pdx1, thus triggering its ubiquitination and proteasomal degradation. However, the underlying mechanisms are not well understood. Here, we present the crystal structure of the SPOP-Pdx1 complex. We show that Pdx1 residues 223-233 bind to SPOP MATH domain with low micromolar affinity. The interface is extended compared to other SPOP-client proteins. Previously, Pdx1 phosphorylation has been proposed to have a regulatory function. In this respect we show that phosphorylation lowers the affinity of Pdx1 to SPOP by isothermal titration calorimetry and nuclear magnetic resonance data. Our data provide insights into a critical protein-protein interaction that regulates cellular Pdx1 levels by SPOP-mediated decay. A reduction of Pdx1 levels in ß cells is linked to apoptosis and considered a hallmark of type 2 diabetes.

Reliability of light microscopy and a computer-assisted replica measurement technique for evaluating the fit of dental copings.

The aim of this in vitro study was to assess the reliability of two measurement systems for evaluating the marginal and internal fit of dental copings. Sixteen CAD/CAM titanium copings were produced for a prepared maxillary canine. To modify the CAD surface model using different parameters (data density; enlargement in different directions), varying fit was created. Five light-body silicone replicas representing the gap between the canine and the coping were made for each coping and for each measurement method: (1) light microscopy measurements (LMMs); and (2) computer-assisted measurements (CASMs) using an optical digitizing system. Two investigators independently measured the marginal and internal fit using both methods. The inter-rater reliability [intraclass correlation coefficient (ICC)] and agreement [Bland-Altman (bias) analyses]: mean of the differences (bias) between two measurements [the closer to zero the mean (bias) is, the higher the agreement between the two measurements] were calculated for several measurement points (marginal-distal, marginal-buccal, axial-buccal, incisal). For the LMM technique, one investigator repeated the measurements to determine repeatability (intra-rater reliability and agreement). For inter-rater reliability, the ICC was 0.848-0.998 for LMMs and 0.945-0.999 for CASMs, depending on the measurement point. Bland-Altman bias was -15.7 to 3.5 µm for LMMs and -3.0 to 1.9 µm for CASMs. For LMMs, the marginal-distal and marginal-buccal measurement points showed the lowest ICC (0.848/0.978) and the highest bias (-15.7 µm/-7.6 µm). With the intra-rater reliability and agreement (repeatability) for LMMs, the ICC was 0.970-0.998 and bias was -1.3 to 2.3 µm. LMMs showed lower interrater reliability and agreement at the marginal measurement points than CASMs, which indicates a more subjective influence with LMMs at these measurement points. The values, however, were still clinically acceptable. LMMs showed very high intra-rater reliability and agreement for all measurement points, indicating high repeatability.

Reliability of light microscopy and a computer-assisted replica measurement technique for evaluating the fit of dental copings

Abstract The aim of this in vitro study was to assess the reliability of two measurement systems for evaluating the marginal and internal fit of dental copings. Material and Methods: Sixteen CAD/CAM titanium copings were produced for a prepared maxillary canine. To modify the CAD surface model using different parameters (data density; enlargement in different directions), varying fit was created. Five light-body silicone replicas representing the gap between the canine and the coping were made for each coping and for each measurement method: (1) light microscopy measurements (LMMs); and (2) computer-assisted measurements (CASMs) using an optical digitizing system. Two investigators independently measured the marginal and internal fit using both methods. The inter-rater reliability [intraclass correlation coefficient (ICC)] and agreement [Bland-Altman (bias) analyses]: mean of the differences (bias) between two measurements [the closer to zero the mean (bias) is, the higher the agreement between the two measurements] were calculated for several measurement points (marginal-distal, marginal-buccal, axial-buccal, incisal). For the LMM technique, one investigator repeated the measurements to determine repeatability (intra-rater reliability and agreement). Results: For inter-rater reliability, the ICC was 0.848-0.998 for LMMs and 0.945-0.999 for CASMs, depending on the measurement point. Bland-Altman bias was −15.7 to 3.5 μm for LMMs and −3.0 to 1.9 μm for CASMs. For LMMs, the marginal-distal and marginal-buccal measurement points showed the lowest ICC (0.848/0.978) and the highest bias (-15.7 μm/-7.6 μm). With the intra-rater reliability and agreement (repeatability) for LMMs, the ICC was 0.970-0.998 and bias was −1.3 to 2.3 μm. Conclusion: LMMs showed lower interrater reliability and agreement at the marginal measurement points than CASMs, which indicates a more subjective influence with LMMs at these measurement points. The values, however, were still clinically acceptable. LMMs showed very high intra-rater reliability and agreement for all measurement points, indicating high repeatability.

Comparison of an analog and digital quantitative and qualitative analysis for the fit of dental copings.

BACKGROUND: Precision in fit is crucial for dental crowns and bridges. Most analyses of fit are based on analog 2D techniques. Aim of this in-vitro study was to compare an analog and a digital quantitative and qualitative analysis for the fit of CAD/CAM fabricated dental copings. METHODS: A prepared steel canine served as master die. CAD surface models, varying in data density, were purposely enlarged in height (Ez), circumference (Exy) and both of these aspects at once (Exyz). Two titanium copings for each variation were produced. The silicone-replica-technique was applied to analyze the fit by means of a 2D analog light microscope measurement (LMM) and a 3D computer-assisted measurement using an optical digitizing system (ODKM97), respectively. RESULTS: In most cases, restorations based on the low data density showed a better fit than those based on high data density. Original size low density data showed the lowest marginal and axial values in the quantitative 2D analyses (LMM and ODKM97). The 3D measurements (ODKM97) revealed best fit of the low density original size specimens, whereas the Ez specimens showed the highest values. Noticeable variations in fit were detected marginally and axially depending on the specific measurement point (mesial, distal, oral, or buccal) for both measurement systems. DISCUSSION: The analog 2D replica technique revealed a loss of information due to the necessary cutting process. By contrast, the digital computer-based method provided 3D quantitative and qualitative results without data loss over the complete surface.

Patient-specific 12-lead ECG reconstruction from sparse electrodes using independent component analysis.

We propose and evaluate a method of 12-lead electrocardiogram (ECG) reconstruction from a three-lead set. The method makes use of independent component analysis and results in adaptive patient-specific transforms. The required calibration process is short and makes use of a single beat. We apply the method to two sets of leads: leads I, II, V2 and the Frank XYZ leads. Performance is evaluated via percent correlation calculations between reconstructed and original leads from a publicly available database of 549 ECG recordings. Results depict percent correlation exceeding 96% for almost all leads. Adaptability of the method's transform is shown to compensate for changes in signal propagation conditions due to breathing, resulting in reduced variance of reconstruction accuracy across beats. This implies that the method is robust to changes that occur after the time of calibration. Accurate and adaptive reconstruction has the potential to augment the clinical significance of wireless ECG systems since the number of sensor nodes placed on the body is limited and the subject could be mobile.

Reconstructing ECG precordial leads from a reduced lead set using independent component analysis.

In this paper, precordial lead reconstruction from a reduced set of leads is considered. We propose the use of independent component analysis to train patient-specific transforms from a reduced lead set to the six precordial leads of the standard 12-lead electrocardiogram. The proposed approach is applied to a publicly available database comprising 549 ECG recordings of patients with varying cardiovascular conditions. The fidelity of reconstruction is measured using percent correlation between the actual and reconstructed signals following a 30 seconds time lapse. The mean correlation is over 95% with a standard deviation under 12.7% for all reconstructed leads. The results demonstrate the potential of the suggested approach to provide a reliable solution to precordial leads reconstruction.