Search

Penicillin binding proteins (PBPs) are involved in biosynthesis, remodeling and recycling of peptidoglycan (PG) in bacteria. PBP-A from Thermosynechococcus elongatus belongs to a cyanobacterial family of enzymes sharing close structural and phylogenetic proximity to class A β-lactamases. With the long-term aim of converting PBP-A into a β-lactamase by directed evolution, we simulated what may happen when an organism like Escherichia coli acquires such a new PBP and observed growth defect associated with the enzyme activity. To further explore the molecular origins of this harmful effect, we decided to characterize deeper the activity of PBP-A both in vitro and in vivo. We found that PBP-A is an enzyme endowed with DD-carboxypeptidase and DD-endopeptidase activities, featuring high specificity towards muropeptides amidated on the D-iso-glutamyl residue. We also show that a low promiscuous activity on non-amidated peptidoglycan deteriorates E. coli's envelope, which is much higher under acidic conditions where substrate discrimination is mitigated. Besides expanding our knowledge of the biochemical activity of PBP-A, this work also highlights that promiscuity may depend on environmental conditions and how it may hinder rather than promote enzyme evolution in nature or in the laboratory., (© 2024. The Author(s).)

Background: Cystic fibrosis (CF) is commonly characterised by thick respiratory mucus. From diagnosis, people with CF are prescribed daily physiotherapy, including airway clearance techniques (ACTs). ACTs consume a large proportion of treatment time, yet the efficacy and effectiveness of ACTs are poorly understood. This study aimed to evaluate associations between the quality and quantity of ACTs and lung function in children and young people with CF., Methods: Project Fizzyo, a longitudinal observational cohort study in the UK, used remote monitoring with electronic pressure sensors attached to four different commercial ACT devices to record real-time, breath-by-breath pressure data during usual ACTs undertaken at home over 16 months in 145 children. ACTs were categorised either as conformant or not with current ACT recommendations based on breath pressure and length measurements, or as missed treatments if not recorded. Daily, weekly and monthly associations between ACT category and lung function were investigated using linear mixed effects regression models adjusting for clinical confounders., Results: After exclusions, 45 224 ACT treatments (135 individuals) and 21 069 days without treatments (141 individuals) were analysed. The mean±sd age of participants was 10.2±2.9 years. Conformant ACTs (21%) had significantly higher forced expiratory volume in 1 s (FEV 1 ) (mean effect size 0.23 (95% CI 0.19-0.27) FEV 1 % pred per treatment) than non-conformant (79%) or missed treatments. There was no benefit from non-conformant or missed treatments and no significant difference in FEV 1 between them (mean effect size 0.02 (95% CI -0.01-0.05) FEV 1 % pred per treatment)., Conclusions: ACTs are beneficial when performed as recommended, but most people use techniques that do not improve lung function. Work is needed to monitor and improve ACT quality and to increase the proportion of people doing effective airway clearance at home., Competing Interests: Conflicts of interest: G. Davies reports personal UKRI fellowship, UKRI and NIHR grants, speaker honoraria from Chiesi Ltd, and speaker honoraria, consulting fees and participation on a board from Vertex Pharmaceuticals, outside the submitted work. S. Stanojevic reports consulting fees from Chiesi Ltd, speaker fees from Vyaire Medical, participation on a board for Ndd technologies, and leadership role from the European Respiratory Society and the American Thoracic Society, outside the submitted work. The remaining authors have no potential competing interests to disclose., (Copyright ©The authors 2023.)

Background: Clinical outcomes are normally captured less frequently than data from remote technologies, leaving a disparity in volumes of data from these different sources. To align these data, flexible polynomial regression was investigated to estimate personalised trends for a continuous outcome over time., Methods: Using electronic health records, flexible polynomial regression models inclusive of a 1st up to a 4th order were calculated to predict forced expiratory volume in 1 s (FEV 1 ) over time in children with cystic fibrosis. The model with the lowest AIC for each individual was selected as the best fit. The optimal parameters for using flexible polynomials were investigated by comparing the measured FEV 1 values to the values given by the individualised polynomial., Results: There were 8,549 FEV 1 measurements from 267 individuals. For individuals with > 15 measurements (n = 178), the polynomial predictions worked well; however, with < 15 measurements (n = 89), the polynomial models were conditional on the number of measurements and time between measurements. The method was validated using BMI in the same population of children., Conclusion: Flexible polynomials can be used to extrapolate clinical outcome measures at frequent time intervals to align with daily data captured through remote technologies., (© 2023. The Author(s).)

Children and young people with CF (CYPwCF) get advice about using positive expiratory pressure (PEP) or oscillating PEP (OPEP) devices to clear sticky mucus from their lungs. However, little is known about the quantity (number of treatments, breaths, or sets) or quality (breath pressures and lengths) of these daily airway clearance techniques (ACTs) undertaken at home. This study used electronic pressure sensors to record real time breath-by-breath data from 145 CYPwCF (6-16y) during routine ACTs over 2 months. ACT quantity and quality were benchmarked against individual prescriptions and accepted recommendations for device use. In total 742,084 breaths from 9,081 treatments were recorded. Individual CYPwCF maintained consistent patterns of ACT quantity and quality over time. Overall, 60% of CYPwCF did at least half their prescribed treatments, while 27% did fewer than a quarter. About 77% of pre-teens did the right number of daily treatments compared with only 56% of teenagers. CYPwCF usually did the right number of breaths. ACT quality (recommended breath length and pressure) varied between participants and depended on device. Breath pressures, lengths and pressure-length relationships were significantly different between ACT devices. PEP devices encouraged longer breaths with lower pressures, while OPEP devices encouraged shorter breaths with higher pressures. More breaths per treatment were within advised ranges for both pressure and length using PEP (30-31%) than OPEP devices (1-3%). Objective measures of quantity and quality may help to optimise ACT device selection and support CYPwCF to do regular effective ACTs., Competing Interests: Conflict of interest statement All authors declare that they have no conflicts of interest., (Copyright © 2022. Published by Elsevier B.V.)

Survival rates from colorectal cancer (CRC) are drastically higher if the disease is detected and treated earlier. Current screening guidelines involve stool-based tests and colonoscopies, whose acceptability and uptake remains low. Routinely collected blood-based biomarkers may offer a low-cost alternative or aid for detecting CRC. Here we aimed to evaluate the pre-diagnostic and diagnostic value of a wide-range of multimodal biomarkers in the UK Biobank dataset, including sociodemographic, lifestyle, medical, physical, and blood and urine-based measures in detecting CRC. We performed a Cox proportional hazard and a tree-boosting model alongside feature selection methods to determine optimal combination of biomarkers. In addition to the modifiable lifestyle factors of obesity, alcohol consumption and cardiovascular health, we showed that blood-based biomarkers that capture the immune response, lipid profile, liver and kidney function are associated with CRC risk. Following feature selection, the final Cox and tree-boosting models achieved a C-index of 0.67 and an AUC of 0.76 respectively. We show that blood-based biomarkers collected in routine examinations are sensitive to preclinical and clinical CRC. They may provide an additive value and improve diagnostic accuracy of current screening tools at no additional cost and help reduce burden on the healthcare system., (© 2023. The Author(s).)

Bcl-x L is an antiapoptotic mitochondrial trans-membrane protein, which is known to play a crucial role in the survival of tumor cells. The deamidation of Bcl-x L is a pivotal switch that regulates its biological function. The potential impact of deamidation on the structure and dynamics of Bcl-x L is directly linked to the intrinsically disordered region (IDR), which is the main site for post-translational modifications (PTMs). In this study, we explored deamidation-induced conformational changes in Bcl-x L to gain insight into its loss of function by performing microsecond-long molecular dynamics (MD) simulations. MD simulation outcomes showed that the IDR motion and interaction patterns have changed notably upon deamidation. Principal component analysis (PCA) demonstrates significant differences between wild-type and deamidated Bcl-x L and suggests that deamidation affects the structure and dynamics of Bcl-x L . The combination of clustering analysis, H-bond analysis, and PCA revealed changes in conformation, interaction, and dynamics upon deamidation. Differences in contact patterns and essential dynamics that lead to a narrowing in the binding groove (BG) are clear indications of deamidation-induced allosteric effects. In line with previous studies, we show that the IDR plays a very important role in the loss of apoptotic functions of Bcl-x L while providing a unique perspective on the underlying mechanism of Bcl-x L deamidation-induced cell death.

Oral cancer is the most common type of head and neck cancer worldwide, leading to approximately 177,757 deaths every year. When identified at early stages, oral cancers can achieve survival rates of up to 75-90%. However, the majority of the cases are diagnosed at an advanced stage mainly due to the lack of public awareness about oral cancer signs and the delays in referrals to oral cancer specialists. As early detection and treatment remain to be the most effective measures in improving oral cancer outcomes, the development of vision-based adjunctive technologies that can detect oral potentially malignant disorders (OPMDs), which carry a risk of cancer development, present significant opportunities for the oral cancer screening process. In this study, we explored the potential applications of computer vision techniques in the oral cancer domain within the scope of photographic images and investigated the prospects of an automated system for detecting OPMD. Exploiting the advancements in deep learning, a two-stage model was proposed to detect oral lesions with a detector network and classify the detected region into three categories (benign, OPMD, carcinoma) with a second-stage classifier network. Our preliminary results demonstrate the feasibility of deep learning-based approaches for the automated detection and classification of oral lesions in real time. The proposed model offers great potential as a low-cost and non-invasive tool that can support screening processes and improve detection of OPMD.

A predictive computational study was conducted in order to assess the efficiency of electrocyclization reactions of keteniminium salts, in an effort to form a variety of heterocyclic systems, namely, 3-amino(benzo)thiophenes, 3-amino(benzo)furans, 3-aminopyrroles, as well as 3-aminoindoles. A density functional theory (DFT) approach was utilized and the effect of heteroatoms (NMe, O, S) was thoroughly investigated by means of population analysis, QTAIM, NICS, ACID, and local reactivity descriptors (Parr and Fukui functions). The electrocyclization of enamines leading to 3-aminopyrroles was shown to be both kinetically and thermodynamically most favorable. Moreover, the pericyclic nature of the electrocyclizations was confirmed using FMO, QTAIM, NICS, and ACID methods. Additionally, substituent effects were investigated in order to give further insight on the reactivity of heteroatom containing keteniminium systems toward electrocyclization reactions. Finally, computational predictions were experimentally confirmed for a selection of keteniminium systems.

Transient bicyclic aziridinium ions are known to undergo ring-expansion reactions, paving the way to functionalized nitrogen-containing heterocycles. In this study, the regioselectivity observed in the ring-expansion reactions of 1-azoniabicyclo[n.1.0]alkanes was investigated from a computational viewpoint to study the ring-expansion pathways of two bicyclic systems with different ring sizes. Moreover, several nucleophiles leading to different experimental results were investigated. The effect of solvation was taken into account using both explicit and implicit solvent models. This theoretical rationalization provides valuable insight into the observed regioselectivity and may be used as a predictive tool in future studies.

Remarkable progress in the area of asymmetric organocatalysis has been achieved in the last decades. Cinchona alkaloids and their derivatives have emerged as powerful organocatalysts owing to their reactivities leading to high enantioselectivities. The widespread usage of cinchona alkaloids has been attributed to their nontoxicity, ease of use, stability, cost effectiveness, recyclability, and practical utilization in industry. The presence of tunable functional groups enables cinchona alkaloids to catalyze a broad range of reactions. Excellent experimental studies have extensively contributed to this field, and highly selective reactions were catalyzed by cinchona alkaloids and their derivatives. Computational modeling has helped elucidate the mechanistic aspects of cinchona alkaloid catalyzed reactions as well as the origins of the selectivity they induce. These studies have complemented experimental work for the design of more efficient catalysts. This Account presents recent computational studies on cinchona alkaloid catalyzed organic reactions and the theoretical rationalizations behind their effectiveness and ability to induce selectivity. Valuable efforts to investigate the mechanisms of reactions catalyzed by cinchona alkaloids and the key aspects of the catalytic activity of cinchona alkaloids in reactions ranging from pharmaceutical to industrial applications are summarized. Quantum mechanics, particularly density functional theory (DFT), and molecular mechanics, including ONIOM, were used to rationalize experimental findings by providing mechanistic insights into reaction mechanisms. B3LYP with modest basis sets has been used in most of the studies; nonetheless, the energetics have been corrected with higher basis sets as well as functionals parametrized to include dispersion M05-2X, M06-2X, and M06-L and functionals with dispersion corrections. Since cinchona alkaloids catalyze reactions by forming complexes with substrates via hydrogen bonds and long-range interactions, the use of split valence triple-ζ basis sets including diffuse and polarization functions on heavy atoms and polarization functions on hydrogens are recommended. Most of the studies have used the continuum-based models to mimic the condensed phase in which organocatalysts function; in some cases, explicit solvation was shown to yield better quantitative agreement with experimental findings. The conformational behavior of cinchona alkaloids is also highlighted as it is expected to shed light on the origin of selectivity and pave the way to a comprehensive understanding of the catalytic mechanism. The ultimate goal of this Account is to provide an up-to-date overlook on cinchona alkaloid catalyzed chemistry and provide insight for future studies in both experimental and theoretical fields.

Aldosterone-producing adenomas (APAs) vary in phenotype and genotype. Zona glomerulosa (ZG)-like APAs frequently have mutations of an L-type calcium channel (LTCC) CaV1.3. Using a novel antagonist of CaV1.3, compound 8, we investigated the role of CaV1.3 on steroidogenesis in the human adrenocortical cell line, H295R, and in primary human adrenal cells. This investigational drug was compared with the common antihypertensive drug nifedipine, which has 4.5-fold selectivity for the vascular LTCC, CaV1.2, over CaV1.3. In H295R cells transfected with wild-type or mutant CaV1.3 channels, the latter produced more aldosterone than wild-type, which was ameliorated by 100 μM of compound 8. In primary adrenal and non-transfected H295R cells, compound 8 decreased aldosterone production similar to high concentration of nifedipine (100 μM). Selective CaV1.3 blockade may offer a novel way of treating primary hyperaldosteronism, which avoids the vascular side effects of CaV1.2-blockade, and provides targeted treatment for ZG-like APAs with mutations of CaV1.3.