CYP116B5hd, a self-sufficient P450 cytochrome: A dataset of its electronic and geometrical properties

This paper documents the dataset obtained from the Electron Paramagnetic Resonance (EPR) study of the electronic properties of a self-sufficient cytochrome P450, CYP116B5hd, which possesses an interesting catalytic activity for synthetic purposes. In fact, when isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Raw data shown in Famulari et al. (2022) and supplementary data in raw and processed forms (figures) are documented and available in this paper. Additionally, simulations of the experimental data together with simulation scripts based for EasySpin, a widespread MATLAB toolbox for EPR spectral simulations, are provided. The procedure for g-value analysis based on a crystal-field theory is also detailed here, offering an interesting tool for comparison of Fe(III)-heme P450 systems. Due to the catalytic interest of the protein, which has been recently discovered, and the correlation that has been reported between g-values and peroxidase function, both, CW-EPR and HYSCORE spectra and data set of the model CYPBM3hd are also provided. Finally, the materials and methods for enzyme production and purification, sample preparation and experimental and spectroscopic procedures a together with instrumental details are described in detail. The data files and simulation scripts can be found in: https://doi.org/10.5281/zenodo.641862

EPR characterization of the heme domain of a self-sufficient cytochrome P450 (CYP116B5)

CYP116B5 is a self-sufficient cytochrome P450 (CYP450) with interesting catalytic properties for synthetic purposes. When isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Here, by means of continuous wave and advanced EPR techniques, the coordination environment of iron in the isolated CYP116B5 heme domain (CYP116b5hd) is characterized. The ligand-free protein shows the characteristic EPR spectrum of a low-spin (S = 1/2) FeIII-heme with gz = 2.440 ± 0.005, gy = 2.25 ± 0.01, gx = 1.92 ± 0.01]. These g-values reflect an electronic ground state very similar to classical P450 monooxygenases rather than P450 peroxygenases. Binding of imidazole results in g-values very close to the ones reported for CYP152 peroxygenases. The detection of hyperfine interactions through HYperfine Sub-level CORrElation (HYSCORE) Spectroscopy experiments, shows that this is due to a nitrogen-mediated axial coordination. This work adds a piece of experimental evidence to the research, aimed at elucidating the features that distinguish the classical P450 enzymes from peroxygenases. It shows that the electronic environment of heme iron of CYP116B5 in the resting state is similar to the classical P450 monooxygenases. Therefore, it is not the critical factor that confers to CYP116B5hd its peroxygenase-like activity, suggesting a crucial role of the protein matrix. © 2022 The Author

A site-specific intraoperative measurement of bone-to-implant contact during implant insertion: A study on bovine ribs using a computerized implant motor

Background/purpose: The aim of the current in vitro study was to determine if there was a correlation between the integrals (I) of the function cutting resistance/depth, obtained using a computerized implant motor, and the bone-to-implant contact (BIC) percentages of dental implants inserted in bovine ribs. Materials and methods: Segments of bovine ribs were used, and a total of 21 perforations were performed. A total of 21 dental implants were inserted in the prepared bone sites. A computerized implant motor ("Torque Measuring Motor") was used to assess, before implant insertion, the values of the bone cutting resistance. The data of bone density obtained by the implant motor were statistically correlated with the BIC percentages. Results: A significant positive linear correlation was found between the integrals measured by the implant motor and the BIC assessed by histomorphometry (r = 0.78, n = 21, P < 0.0001). Indeed, the increase of the integral values recorded by the reader matched with the increase of BIC percentages measured by histomorphometry. Pearson correlation coefficient for linear regression (R2) between values assessed by the surgical motor and histomorphometry was 0.61 (P < 0.0001), indicating that 61% of the data points were aligned with the regression line. Conclusion: The instrument under testing seems to provide a reliable quantitative estimator, the integral, of the final BIC achieved at implant insertion, and therefore of the implant primary stability, and could represent a significant aid for a proper planning of rehabilitations with the use of dental implants

Human cytochrome P450 3A4 as a biocatalyst: Effects of the engineered linker in modulation of coupling efficiency in 3A4-BMR chimeras

Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from Bacillus megaterium BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a V(max) increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome c reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis

A New Algorithm for The Surgical Management of Defects of the Scalp

Scalp reconstruction is a daily challenge for plastic surgeons. The authors propose their algorithm for reconstructive surgery after ablative surgery. They considered not only the size defect but also the anatomical defect and the clinical condition of the patient to achieve the best choice for reconstruction. During the two-year period, a total of 86 procedures were performed on 78 patients. We used five different techniques for reconstruction, including primary closure, graft, local or free flap, and a dermal regeneration template. No statistical difference of complication was observed in the different groups. We consider our algorithm a useful improvement in the management of the defect at the vertex.</jats:p

Social Perception of Reconstruction following Orbital Exenteration

Background: Orbital exenteration, the removal of the entire globe, eyelids, and orbital content, is indicated in extensive neoplastic disease involving the orbital region. Although a functional reconstruction of orbital exenteration defects is mandatory, aesthetic concerns need to be considered. Facial disfigurement following reconstructive surgery often leads to great discomfort and social retirement, which can limit social interaction. The aim of this study was to explore how the society perceives the aspect of patients who underwent orbital exenteration and subsequent reconstruction, comparing two different types of reconstruction: standard anterolateral thigh (ALT) or "sandwich" fascial ALT (SALT) free flap. Methods: An online survey was created based on four questions regarding the perception of reconstruction (discomfort at looking at that patient, perception of unhealthiness, hypothesis of social life impairment, etc); five possible answers were provided, ranging from "completely" to "not at all." The survey was administered to the general population and to medical students. Results: In total, 255 people participated to the survey (130 medical students and 125 people of the general population); a total of 245 surveys were considered eligible (10 were incomplete and then discharged). Statistical significance was found (P &lt; 0.001) regarding the better overall appearance of the SALT group over the ALT one. Conclusions: After analysis, the surgical outcome after SALT reconstruction has been found to be less disruptive in both groups, due to a reduced scar burden and a more pleasant orbital pocket. Our results encourage more research in the field of postexenteration reconstruction to achieve more aesthetic and social acceptability

Gut Microbiota as a Target for Preventive and Therapeutic Intervention against Food Allergy

The gut microbiota plays a pivotal role in immune system development and function. Modification in the gut microbiota composition (dysbiosis) early in life is a critical factor affecting the development of food allergy. Many environmental factors including caesarean delivery, lack of breast milk, drugs, antiseptic agents, and a low-fiber/high-fat diet can induce gut microbiota dysbiosis, and have been associated with the occurrence of food allergy. New technologies and experimental tools have provided information regarding the importance of select bacteria on immune tolerance mechanisms. Short-chain fatty acids are crucial metabolic products of gut microbiota responsible for many protective effects against food allergy. These compounds are involved in epigenetic regulation of the immune system. These evidences provide a foundation for developing innovative strategies to prevent and treat food allergy. Here, we present an overview on the potential role of gut microbiota as the target of intervention against food allergy

Ranolazine Attenuates Trastuzumab-Induced Heart Dysfunction by Modulating ROS Production

The ErbB2 blocker trastuzumab improves survival in oncologic patients, but can cause cardiotoxicity. The late Na+ current inhibitor ranolazine has been shown to counter experimental HF, including doxorubicin cardiotoxicity (a condition characterized by derangements in redox balance), by lowering the levels of reactive oxygen species (ROS). Since ErbB2 can modulate ROS signaling, we tested whether trastuzumab cardiotoxicity could be blunted by ranolazine via redox-mediated mechanisms. Trastuzumab decreased fractional shortening and ejection fraction in mice, but ranolazine prevented heart dysfunction when co-administered with trastuzumab. Trastuzumab cardiotoxicity was accompanied by elevations in natriuretic peptides and matrix metalloproteinase 2 (MMP2) mRNAs, which were not elevated with co-treatment with ranolazine. Trastuzumab also increased cleavage of caspase-3, indicating activation of the proapoptotic machinery. Again, ranolazine prevented this activation. Interestingly, Neonatal Rat Ventricular Myocytes (NRVMs), labeled with MitoTracker Red and treated with trastuzumab, showed only a small increase in ROS compared to baseline conditions. We then stressed trastuzumab-treated cells with the beta-agonist isoproterenol to increase workload, and we observed a significant increase of probe fluorescence, compared with cells treated with isoproterenol alone, reflecting induction of oxidative stress. These effects were blunted by ranolazine, supporting a role for INa inhibition in the regulation of redox balance also in trastuzumab cardiotoxicity

BIOTEX-biosensing textiles for personalised healthcare management.

Textile-based sensors offer an unobtrusive method of continually monitoring physiological parameters during daily activities. Chemical analysis of body fluids, noninvasively, is a novel and exciting area of personalized wearable healthcare systems. BIOTEX was an EU-funded project that aimed to develop textile sensors to measure physiological parameters and the chemical composition of body fluids, with a particular interest in sweat. A wearable sensing system has been developed that integrates a textile-based fluid handling system for sample collection and transport with a number of sensors including sodium, conductivity, and pH sensors. Sensors for sweat rate, ECG, respiration, and blood oxygenation were also developed. For the first time, it has been possible to monitor a number of physiological parameters together with sweat composition in real time. This has been carried out via a network of wearable sensors distributed around the body of a subject user. This has huge implications for the field of sports and human performance and opens a whole new field of research in the clinical setting