PPIscreenML: Structure-based screening for protein-protein interactions using AlphaFold.

Protein-protein interactions underlie nearly all cellular processes. With the advent of protein structure prediction methods such as AlphaFold2 (AF2), models of specific protein pairs can be built extremely accurately in most cases. However, determining the relevance of a given protein pair remains an open question. It is presently unclear how to use best structure-based tools to infer whether a pair of candidate proteins indeed interact with one another: ideally, one might even use such information to screen amongst candidate pairings to build up protein interaction networks. Whereas methods for evaluating quality of modeled protein complexes have been co-opted for determining which pairings interact (e.g., pDockQ and iPTM), there have been no rigorously benchmarked methods for this task. Here we introduce PPIscreenML, a classification model trained to distinguish AF2 models of interacting protein pairs from AF2 models of compelling decoy pairings. We find that PPIscreenML out-performs methods such as pDockQ and iPTM for this task, and further that PPIscreenML exhibits impressive performance when identifying which ligand/receptor pairings engage one another across the structurally conserved tumor necrosis factor superfamily (TNFSF). Analysis of benchmark results using complexes not seen in PPIscreenML development strongly suggest that the model generalizes beyond training data, making it broadly applicable for identifying new protein complexes based on structural models built with AF2.

A Descriptor Set for Quantitative Structure-property Relationship Prediction in Biologics.

There has been a remarkable increase in the number of biologics, especially monoclonal antibodies, in the market over the last decade. In addition to attaining the desired binding to their targets, a crucial aspect is the 'developability' of these drugs, which includes several desirable properties such as high solubility, low viscosity and aggregation, physico-chemical stability, low immunogenicity and low poly-specificity. The lack of any of these desirable properties can lead to significant hurdles in advancing them to the clinic and are often discovered only during late stages of drug development. Hence, in silico methods for early detection of these properties, particularly the ones that affect aggregation and solubility in the earlier stages can be highly beneficial. We have developed a computational framework based on a large and diverse set of protein specific descriptors that is ideal for making liability predictions using a QSPR (quantitative structure-property relationship) approach. This set offers a high degree of feature diversity that may coarsely be classified based on (1) sequence (2) structure and (3) surface patches. We assess the sensitivity and applicability of these descriptors in four dedicated case studies that are believed to be representative of biophysical characterizations commonly employed during the development process of a biologics drug candidate. In addition to data sets obtained from public sources, we have validated the descriptors on novel experimental data sets in order to address antibody developability and to generate prospective predictions on Adnectins. The results show that the descriptors are well suited to assist in the improvement of protein properties of systems that exhibit poor solubility or aggregation.

Accurate Prediction of Protein Thermodynamic Stability Changes upon Residue Mutation using Free Energy Perturbation.

This work describes the application of a physics-based computational approach to predict the relative thermodynamic stability of protein variants, and evaluates the quantitative accuracy of those predictions compared to experimental data obtained from a diverse set of protein systems assayed at variable pH conditions. Physical stability is a key determinant of the clinical and commercial success of biological therapeutics, vaccines, diagnostics, enzymes and other protein-based products. Although experimental techniques for measuring the impact of amino acid residue mutation on the stability of proteins exist, they tend to be time consuming and costly, hence the need for accurate prediction methods. In contrast to many of the commonly available computational methods for stability prediction, the Free Energy Perturbation approach applied in this paper explicitly accounts for solvent effects and samples conformational dynamics using a rigorous molecular dynamics simulation process. On the entire validation dataset, consisting of 328 single point mutations spread across 14 distinct protein structures, our results show good overall correlation with experiment with an R2 of 0.65 and a low mean unsigned error of 0.95 kcal/mol. Application of the FEP approach in conjunction with experimental assessment techniques offers opportunities to lower the time and expense of product development and reduce the risk of costly late-stage failures.

Long-term switching of single photochromic triads based on dithienylcyclopentene and fluorophores at cryogenic temperatures.

Photochromic molecules can be reversibly converted between two bistable forms by light. These systems have been intensively studied for applications as molecular memories, sensing devices, or super-resolution optical microscopy. Here, we study the long-term switching behavior of single photochromic triads under oxygen-free conditions at 10 K. The triads consist of a photochromic unit that is covalently linked to two strong fluorophores that were employed for monitoring the light-induced conversions of the switch via changes in the fluorescence intensity from the fluorophores. As dyes we use either perylene bisimide or boron-dipyrromethen, and as photochromic switch we use dithienylcyclopentene (DCP). Both types of triads showed high fatigue resistance allowing for up to 6000 switching cycles of a single triad corresponding to time durations in the order of 80 min without deterioration. Long-term analysis of the switching cycles reveals that the probability that an intensity change in the emission from the dyes can be assigned to an externally stimulated conversion of the DCP (rather than to stochastic blinking of the dye molecules) amounts to 0.7 ± 0.1 for both types of triads. This number is far too low for optical data storage using single triads and implications concerning the miniaturization of optical memories based on such systems will be discussed. Yet, together with the high fatigue resistance, this number is encouraging for applications in super-resolution optical microscopy on frozen biological samples.

MRI for the display of autologous onlay bone grafts during early healing-an experimental study.

OBJECTIVES: Autologous bone grafts are the gold standard to augment deficient alveolar bone. Dimensional graft alterations during healing are not known as they are not accessible to radiography. Therefore, MRI was used to display autologous onlay bone grafts in vivo during early healing. METHODS AND MATERIALS: Ten patients with alveolar bone atrophy and autologous onlay grafts were included. MRI was performed with a clinical MR system and an intraoral coil preoperatively (t0), 1 week (t1), 6 weeks (t2) and 12 weeks (t3) postoperatively, respectively. The graft volumes were assessed in MRI by manual segmentation by three examiners. Graft volumes for each time point were calculated and dimensional alteration was documented. Cortical and cancellous proportions of bone grafts were assessed. The intraobserver and interobserver variability were calculated. Statistical analysis was performed using a mixed linear regression model. RESULTS: Autologous onlay bone grafts with cortical and cancellous properties were displayed in vivo in eight patients over 12 weeks. The fixation screws were visible as signal voids with a thin hyperintense fringe. The calculated volumes were between 0.12-0.74 cm3 (t1), 0.15-0.73 cm3 (t2), and 0.17-0.64 cm3 (t3). Median changes of bone graft volumes of -15% were observed. There was no significant difference between the examiners (p = 0.3). CONCLUSIONS: MRI is eligible for the display and longitudinal observation of autologous onlay bone grafts. Image artifacts caused measurements deviations in some cases and minimized the precise assessment of graft volume. To the knowledge of the authors, this is the first study that used MRI for the longitudinal observation of autologous onlay bone grafts.

Optically tracked and 3D printed haptic phantom hand for surgical training system.

BACKGROUND: For surgical fixation of bone fractures of the human hand, so-called Kirschner-wires (K-wires) are drilled through bone fragments. Due to the minimally invasive drilling procedures without a view of risk structures like vessels and nerves, a thorough training of young surgeons is necessary. For the development of a virtual reality (VR) based training system, a three-dimensional (3D) printed phantom hand is required. To ensure an intuitive operation, this phantom hand has to be realistic in both, its position relative to the driller as well as in its haptic features. The softest 3D printing material available on the market, however, is too hard to imitate human soft tissue. Therefore, a support-material (SUP) filled metamaterial is used to soften the raw material. Realistic haptic features are important to palpate protrusions of the bone to determine the drilling starting point and angle. An optical real-time tracking is used to transfer position and rotation to the training system. METHODS: A metamaterial already developed in previous work is further improved by use of a new unit cell. Thus, the amount of SUP within the volume can be increased and the tissue is softened further. In addition, the human anatomy is transferred to the entire hand model. A subcutaneous fat layer and penetration of air through pores into the volume simulate shiftability of skin layers. For optical tracking, a rotationally symmetrical marker attached to the phantom hand with corresponding reference marker is developed. In order to ensure trouble-free position transmission, various types of marker point applications are tested. RESULTS: Several cuboid and forearm sample prints lead to a final 30 centimeter long hand model. The whole haptic phantom could be printed faultless within about 17 hours. The metamaterial consisting of the new unit cell results in an increased SUP share of 4.32%. Validated by an expert surgeon study, this allows in combination with a displacement of the uppermost skin layer a good palpability of the bones. Tracking of the hand marker in dodecahedron design works trouble-free in conjunction with a reference marker attached to the worktop of the training system. CONCLUSIONS: In this work, an optically tracked and haptically correct phantom hand was developed using dual-material 3D printing, which can be easily integrated into a surgical training system.

Quantitative Analysis of Surface Contouring with Pulsed Bipolar Radiofrequency on Thin Chondromalacic Cartilage.

The purpose of this study was to evaluate the quality of surface contouring of chondromalacic cartilage by bipolar radio frequency energy using different treatment patterns in an animal model, as well as examining the impact of the treatment onto chondrocyte viability by two different methods. Our experiments were conducted on 36 fresh osteochondral sections from the tibia plateau of slaughtered 6-month-old pigs, where the thickness of the cartilage is similar to that of human wrist cartilage. An area of 1 cm2 was first treated with emery paper to simulate the chondromalacic cartilage. Then, the treatment with RFE followed in 6 different patterns. The osteochondral sections were assessed for cellular viability (live/dead assay, caspase (cell apoptosis marker) staining, and quantitative analysed images obtained by fluorescent microscopy). For a quantitative characterization of none or treated cartilage surfaces, various roughness parameters were measured using confocal laser scanning microscopy (Olympus LEXT OLS 4000 3D). To describe the roughness, the Root-Mean-Square parameter (Sq) was calculated. A smoothing effect of the cartilage surface was detectable upon each pattern of RFE treatment. The Sq for native cartilage was Sq = 3.8 ± 1.1 µm. The best smoothing pattern was seen for two RFE passes and a 2-second pulsed mode (B2p2) with an Sq = 27.3 ± 4.9 µm. However, with increased smoothing, an augmentation in chondrocyte death up to 95% was detected. Using bipolar RFE treatment in arthroscopy for small joints like the wrist or MCP joints should be used with caution. In the case of chondroplasty, there is a high chance to destroy the joint cartilage.

Force-feedback assisted and virtual fixtures based K-wire drilling simulation.

One common method to fix fractures of the human hand after an accident is an osteosynthesis with Kirschner wires (K-wires) to stabilize the bone fragments. The insertion of K-wires is a delicate minimally invasive surgery, because surgeons operate almost without a sight. Since realistic training methods are time consuming, costly and insufficient, a virtual-reality (VR) based training system for the placement of K-wires was developed. As part of this, the current work deals with the real-time bone drilling simulation using a haptic force-feedback device. To simulate the drilling, we introduce a virtual fixture based force-feedback drilling approach. By decomposition of the drilling task into individual phases, each phase can be handled individually to perfectly control the drilling procedure. We report about the related finite state machine (FSM), describe the haptic feedback of each state and explain, how to avoid jerking of the haptic force-feedback during state transition. The usage of the virtual fixture approach results in a good haptic performance and a stable drilling behavior. This was confirmed by 26 expert surgeons, who evaluated the virtual drilling on the simulator and rated it as very realistic. To make the system even more convincing, we determined real drilling feed rates through experimental pig bone drilling and transferred them to our system. Due to a constant simulation thread we can guarantee a precise drilling motion. Virtual fixtures based force-feedback calculation is able to simulate force-feedback assisted bone drilling with high quality and, thus, will have a great potential in developing medical applications.

Imitating human soft tissue on basis of a dual-material 3D print using a support-filled metamaterial to provide bimanual haptic for a hand surgery training system.

BACKGROUND: Currently, it is common practice to use three-dimensional (3D) printers not only for rapid prototyping in the industry, but also in the medical area to create medical applications for training inexperienced surgeons. In a clinical training simulator for minimally invasive bone drilling to fix hand fractures with Kirschner-wires (K-wires), a 3D-printed hand phantom must not only be geometrically but also haptically correct. Due to a limited view during an operation, surgeons need to perfectly localize underlying risk structures only by feeling of specific bony protrusions of the human hand. METHODS: The goal of this experiment is to imitate human soft tissue with its haptic and elasticity for a realistic hand phantom fabrication, using only a dual-material 3D printer and support-material-filled metamaterial between skin and bone. We present our workflow to generate lattice structures between hard bone and soft skin with iterative cube edge (CE) or cube face (CF) unit cells. Cuboid and finger shaped sample prints with and without inner hard bone in different lattice thickness are constructed and 3D printed. RESULTS: The most elastic available rubber-like material is too firm to imitate soft tissue. By reducing the amount of rubber in the inner volume through support material (SUP), objects become significantly softer. Without metamaterial, after disintegration, the SUP can be shifted through the volume and thus the body loses its original shape. Although the CE design increases the elasticity, it cannot restore the fabric form. In contrast to CE, the CF design increases not only the elasticity but also guarantees a local limitation of the SUP. Therefore, the body retains its shape and internal bones remain in its intended place. Various unit cell sizes, lattice thickening and skin thickness regulate the rubber material and SUP ratio. Test prints with higher SUP and lower rubber material percentage appear softer and vice versa. This was confirmed by an expert surgeon evaluation. Subjects adjudged pure rubber-like material as too firm and samples only filled with SUP or lattice structure in CE design as not suitable for imitating tissue. 3D-printed finger samples in CF design were rated as realistic compared to the haptic of human tissue with a good palpable bone structure. CONCLUSIONS: We developed a new dual-material 3D print technique to imitate soft tissue of the human hand with its haptic properties. Blowy SUP is trapped within a lattice structure to soften rubber-like 3D print material, which makes it possible to reproduce a realistic replica of human hand soft tissue.

AggScore: Prediction of aggregation-prone regions in proteins based on the distribution of surface patches.

Protein aggregation is a phenomenon that has attracted considerable attention within the pharmaceutical industry from both a developability standpoint (to ensure stability of protein formulations) and from a research perspective for neurodegenerative diseases. Experimental identification of aggregation behavior in proteins can be expensive; and hence, the development of accurate computational approaches is crucial. The existing methods for predicting protein aggregation rely mostly on the primary sequence and are typically trained on amyloid-like proteins. However, the training bias toward beta amyloid peptides may worsen prediction accuracy of such models when applied to larger protein systems. Here, we present a novel algorithm to identify aggregation-prone regions in proteins termed "AggScore" that is based entirely on three-dimensional structure input. The method uses the distribution of hydrophobic and electrostatic patches on the surface of the protein, factoring in the intensity and relative orientation of the respective surface patches into an aggregation propensity function that has been trained on a benchmark set of 31 adnectin proteins. AggScore can accurately identify aggregation-prone regions in several well-studied proteins and also reliably predict changes in aggregation behavior upon residue mutation. The method is agnostic to an amyloid-specific aggregation context and thus may be applied to globular proteins, small peptides and antibodies.

Design and Application of 6mA-Specific Zinc-Finger Proteins for the Readout of DNA Methylation.

Designed zinc-finger (ZnF) proteins can recognize AT base pairs by H-bonds in the major groove, which are disrupted, if the adenine base is methylated at the N6 position. Based on this principle, we have recently designed a ZnF protein, which does not bind to DNA, if its recognition site is methylated. In this review, we summarize the principles of the recognition of methylated DNA by proteins and describe the design steps starting with the initial bacterial two-hybrid screening of three-domain ZnF proteins that do not bind to CcrM methylated target sites, followed by their di- and tetramerization to improve binding affinity and specificity. One of the 6mA-specific ZnF proteins was used as repressor to generate a methylation-sensitive promoter/repressor system. This artificial promoter/repressor system was employed to regulate the expression of a CcrM DNA methyltransferase gene, thereby generating an epigenetic system with positive feedback, which can exist in two stable states, an off-state with unmethylated promoter, bound ZnF and repressed gene expression, and an on-state with methylated promoter and active gene expression. This system can memorize transient signals approaching bacterial cells and store the input in the form of DNA methylation patterns. More generally, the ability to bind to DNA in a methylation-dependent manner gives ZnF and TAL proteins an advantage over CRISPR/Cas as DNA-targeting device by allowing methylation-dependent genome or epigenome editing.

3D analysis of Osteosyntheses material using semi-automated CT segmentation: a case series of a 4 corner fusion plate.

BACKROUND: Scaphoidectomy and midcarpal fusion can be performed using traditional fixation methods like K-wires, staples, screws or different dorsal (non)locking arthrodesis systems. The aim of this study is to test the Aptus four corner locking plate and to compare the clinical findings to the data revealed by CT scans and semi-automated segmentation. METHODS: This is a retrospective review of eleven patients suffering from scapholunate advanced collapse (SLAC) or scaphoid non-union advanced collapse (SNAC) wrist, who received a four corner fusion between August 2011 and July 2014. The clinical evaluation consisted of measuring the range of motion (ROM), strength and pain on a visual analogue scale (VAS). Additionally, the Disabilities of the Arm, Shoulder and Hand (QuickDASH) and the Mayo Wrist Score were assessed. A computerized tomography (CT) of the wrist was obtained six weeks postoperatively. After semi-automated segmentation of the CT scans, the models were post processed and surveyed. RESULTS: During the six-month follow-up mean range of motion (ROM) of the operated wrist was 60°, consisting of 30° extension and 30° flexion. While pain levels decreased significantly, 54% of grip strength and 89% of pinch strength were preserved compared to the contralateral healthy wrist. Union could be detected in all CT scans of the wrist. While X-ray pictures obtained postoperatively revealed no pathology, two user related technical complications were found through the 3D analysis, which correlated to the clinical outcome. CONCLUSION: Due to semi-automated segmentation and 3D analysis it has been proved that the plate design can keep up to the manufacturers' promises. Over all, this case series confirmed that the plate can compete with the coexisting techniques concerning clinical outcome, union and complication rate.

Temperature dependence of the conversion efficiency of photochromic perylene bisimide dithienylcyclopentene triads embedded in a polymer.

Photochromic molecules that are covalently linked to a strong fluorophore combine the requirements of external control and strong fluorescence, which will become increasingly important for super-resolution microscopy techniques based on single molecules. However, given the bulky structure of such constructs, steric hindrance might affect their photoconversion efficiencies upon immobilising them for imaging purposes. In this study the efficiencies of the photochromic conversion processes of molecular triads that are embedded in a polymer have been studied as a function of temperature. The triads consist of two perylene bisimide dye molecules that are connected via a dithienylcyclopentene photochromic bridge that undergoes a cyclization/cycloreversion reaction upon appropriate illumination. It is found that photochromic switching remains active, even at 5 K, yet with reduced but finite efficiency for the cycloreversion reaction. This might even be an advantage for the achievement of high labelling densities in super-resolution microscopy.

Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation.

Epigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology. Here, we designed synthetic epigenetic memory systems using DNA methylation sensitive engineered zinc finger proteins to repress a memory operon comprising the CcrM methyltransferase and a reporter. Triggering by heat, nutrients, ultraviolet irradiation or DNA damaging compounds induces CcrM expression and DNA methylation. In the induced on-state, methylation in the operator of the memory operon prevents zinc finger protein binding leading to positive feedback and permanent activation. Using an mf-Lon protease degradable CcrM variant enables reversible switching. Epigenetic memory systems have numerous potential applications in synthetic biology, including life biosensors, death switches or induction systems for industrial protein production. The large variety of bacterial DNA methyltransferases potentially allows for massive multiplexing of signal storage and logical operations depending on more than one input signal.

Deliberate Switching of Single Photochromic Triads.

Photochromic molecules can be reversibly converted between two bistable conformations by light, and are considered as promising building blocks in novel macromolecular structures for sensing and imaging techniques. We have studied individual molecular triads consisting of two strong fluorophores (perylene bisimide) that are covalently linked via a photochromic unit (dithienylcyclopentene) and distinguished between deliberate switching and spontaneous blinking. It was verified that the probability for observing deliberate light-induced switching of a single triad (rather than stochastic blinking) amounts to 0.8 ± 0.1. In a few exceptional cases this probability can exceed 0.95. These numbers are sufficiently large for application in sensitive biosensing, and super-resolution imaging. This opens the possibility to develop devices that can be controlled by an external optical stimulus on a truly molecular length scale.

In Situ Atom Probe Deintercalation of Lithium-Manganese-Oxide.

Atom probe tomography is routinely used for the characterization of materials microstructures, usually assuming that the microstructure is unaltered by the analysis. When analyzing ionic conductors, however, gradients in the chemical potential and the electric field penetrating dielectric atom probe specimens can cause significant ionic mobility. Although ionic mobility is undesirable when aiming for materials characterization, it offers a strategy to manipulate materials directly in situ in the atom probe. Here, we present experimental results on the analysis of the ionic conductor lithium-manganese-oxide with different atom probe techniques. We demonstrate that, at a temperature of 30 K, characterization of the materials microstructure is possible without measurable Li mobility. Also, we show that at 298 K the material can be deintercalated, in situ in the atom probe, without changing the manganese-oxide host structure. Combining in situ atom probe deintercalation and subsequent conventional characterization, we demonstrate a new methodological approach to study ionic conductors even in early stages of deintercalation.

Simultaneous integrated boost (SIB) radiation therapy of right sided breast cancer with and without flattening filter - A treatment planning study.

BACKGROUND: The aim of the study was to compare the two irradiation modes with (FF) and without flattening filter (FFF) for three different treatment techniques for simultaneous integrated boost radiation therapy of patients with right sided breast cancer. METHODS: An Elekta Synergy linac with Agility collimating device is used to simulate the treatment of 10 patients. Six plans were generated in Monaco 5.0 for each patient treating the whole breast and a simultaneous integrated boost (SIB) volume: intensity modulated radiation therapy (IMRT), volumetric modulated arc therapy (VMAT) and a tangential arc VMAT (tVMAT), each with and without flattening filter. Plan quality was assessed considering target coverage, sparing of the contralateral breast, the lungs, the heart and the normal tissue. All plans were verified by a 2D-ionisation-chamber-array and delivery times were measured and compared. The Wilcoxon test was used for statistical analysis with a significance level of 0.05. RESULTS: Significantly best target coverage and homogeneity was achieved using VMAT FFF with V95% = (98.7 ± 0.8) % and HI = (8.2 ± 0.9) % for the SIB and V95% = (98.3 ± 0.7) % for the PTV, whereas tVMAT showed significantly lowest doses to the contralateral organs at risk with a Dmean of (0.7 ± 0.1) Gy for the contralateral lung, (1.0 ± 0.2) Gy for the contralateral breast and (1.4 ± 0.2) Gy for the heart. All plans passed the gamma evaluation with a mean passing rate of (99.2 ± 0.8) %. Delivery times were significantly reduced for VMAT and tVMAT but increased for IMRT, when FFF was used. Lowest delivery times were observed for tVMAT FFF with (1:20 ± 0:07) min. CONCLUSION: Balancing target coverage, OAR sparing and delivery time, VMAT FFF and tVMAT FFF are considered the preferable of the investigated treatment options in simultaneous integrated boost irradiation of right sided breast cancer for the combination of an Elekta Synergy linac with Agility and the treatment planning system Monaco 5.0.

Second Cancer Risk after simultaneous integrated boost radiation therapy of right sided breast cancer with and without flattening filter.

BACKGROUND: The aim of this study was to investigate if the flattening filter free mode (FFF) of a linear accelerator reduces the excess absolute risk (EAR) for second cancer as compared to the flat beam mode (FF) in simultaneous integrated boost (SIB) radiation therapy of right-sided breast cancer. PATIENTS AND METHODS: Six plans were generated treating the whole breast to 50.4 Gy and a SIB volume to 63 Gy on CT data of 10 patients: intensity-modulated radiation therapy (IMRT), volumetric modulated arc therapy (VMAT), and a tangential arc VMAT (tVMAT), each with flattening filter and without. The EAR was calculated for the contralateral breast and the lungs from dose-volume histograms (DVH) based on the linear-exponential, the plateau, and the full mechanistic dose-response model. Peripheral low-dose measurements were performed to compare the EAR in more distant regions as the thyroids and the uterus. RESULTS: FFF reduces the EAR significantly in the contralateral and peripheral organs for tVMAT and in the peripheral organs for VMAT. No reduction was found for IMRT. The lowest EAR for the contralateral breast and lung was achieved with tVMAT FFF, reducing the EAR by 25 % and 29 % as compared to tVMAT FF, and by 44 % to 58 % as compared to VMAT and IMRT in both irradiation modes. tVMAT FFF showed also the lowest peripheral dose corresponding to the lowest EAR in the thyroids and the uterus. CONCLUSION: The use of FFF mode allows reducing the EAR significantly when tVMAT is used as the treatment technique. When second cancer risk is a major concern, tVMAT FFF is considered the preferred treatment option in SIB irradiation of right-sided breast cancer.

Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM.

CcrM-related DNA-(adenine N6)-methyltransferases play very important roles in the biology of Caulobacter crescentus and other alpha-proteobacteria. These enzymes methylate GANTC sequences, but the molecular mechanism by which they recognize their target sequence is unknown. We carried out multiple sequence alignments and noticed that CcrM enzymes contain a conserved C-terminal domain (CTD) which is not present in other DNA-(adenine N6)-methyltransferases and we show here that deletion of this part abrogates catalytic activity and DNA binding of CcrM. A mutational study identified 7 conserved residues in the CTD (out of 13 tested), mutation of which led to a strong reduction in catalytic activity. All of these mutants showed altered DNA binding, but no change in AdoMet binding and secondary structure. Some mutants exhibited reduced DNA binding, but others showed an enhanced DNA binding. Moreover, we show that CcrM does not specifically bind to DNA containing GANTC sequences. Taken together, these findings suggest that the specific CcrM-DNA complex undergoes a conformational change, which is endergonic but essential for catalytic activity and this step is blocked by some of the mutations. Moreover, our data indicate that the CTD of CcrM is involved in DNA binding and recognition. This suggests that the CTD functions as target recognition domain of CcrM and, therefore, CcrM can be considered the first example of a δ-type DNA-(adenine N6)-methyltransferase identified so far.

Association between left atrial low-voltage area, serum apoptosis, and fibrosis biomarkers and incidence of silent cerebral events after catheter ablation of atrial fibrillation.

BACKGROUND: Silent cerebral events (SCE) have been identified on cerebral diffusion-weighted cerebral magnetic resonance imaging (DE-MRI) after catheter ablation (CA) of atrial fibrillation (AF). The purpose of this study was to investigate the impact of atrial remodeling on the incidence of SCE after AF CA. METHODS: Forty patients (67.8 ± 10 years, 47.5 % women) with symptomatic paroxysmal (n = 11, 27.5 %) or persistent AF undergoing AF CA were prospectively enrolled. LA fibrosis was estimated by intraprocedural bipolar voltage mapping in sinus rhythm. Apoptosis-stimulating fragment (Fas-Ligand) and amino terminal peptide from collagen III (PIIINP) concentrations were analyzed of LA and femoral vein blood. Cerebral DE-MRI was performed 1 to 2 days after CA of AF for detection of SCE. In nine patients (22.5 %), new SCE were detected on DE-MRI after AF CA. RESULTS: Patients with SCE had higher CHA2DS2-VASc score, larger left atrial diameter (LADmax), and higher surface area of left atrial low-voltage (24 ± 11.2 vs 3.5 ± 4.2 %, p < 0.0001). Concentrations of peripheral PIIINP (103.7 ± 25.9 vs 81.8 ± 16.7 pg/ml, p < 0.01) and Fas-Ligand (124.1 ± 22.4 vs 87.6 ± 19.4 pg/ml, p < 0.01) were significantly higher in patients with SCE and correlated to low-voltage surface area (p < 0.01). Multivariable logistic regression analysis revealed peripheral Fas-Ligand, LADmax, CHA2DS2-Vasc score, and LA low-voltage area proportion to be independent predictors for the development of SCE. CONCLUSIONS: LA remodeling, estimated by LADmax and LA low-voltage area, has significant relationship with the risk of SCE after AF ablation. Moreover, Fas-Ligand may act as an independent predictor for SCE in the context of AF CA.