Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation

In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC)

The art and science of priority-setting: assessing the value of Public Health England’s Prioritization Framework

Background Findings are presented from the evaluation of Public Health England’s (PHE) Prioritization Framework (PF) aimed to assist local authority commissioners with their public health investment and disinvestment decisions. The study explored the take up of the PF in three early adopter local authority settings. Methods Semi-structured interviews (n = 30) across three local authorities supplemented by participant observation of workshops. Results Participants acknowledged that the PF provided a systematic means of guiding priority-setting and one that encouraged transparency over investment and disinvestment decisions. The role performed by PHE and its regional teams in facilitating the process was especially welcomed and considered critical to the adoption process. However, uptake of the PF required a significant investment of time and commitment from public health teams at a time when resources were stretched. The impact of the political environment in the local government was a major factor determining the likely uptake of the PF. Ensuring committed leadership and engagement from senior politicians and officers was regarded as critical to success. Conclusions The study assessed the value and impact of PHE’s PF tool in three early adopter local authorities. Further research could explore the value of the tool in aiding investment and disinvestment decisions and its impact on spending

Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysi

CCD time-series photometry of the globular cluster NGC 5053: RR Lyrae, Blue Stragglers and SX Phoenicis stars revisited

We report the results of CCD $V$, $r$ and $I$ time-series photometry of the globular cluster NGC 5053. New times of maximum light are given for the eight known RR Lyrae stars in the field of our images and their periods are revised. Their $V$ light curves were Fourier decomposed to estimate their physical parameters. A discussion on the accuracy of the Fourier-based iron abundances, temperatures, masses and radii is given. New periods are found for the 5 known SX Phe stars and a critical discussion of their secular period changes is offered. The mean iron abundance for the RR Lyrae stars is found to be [Fe/H] $\sim -1.97 \pm 0.16$ and lower values are not supported by the present analysis. The absolute magnitude calibrations of the RR Lyrae stars yield an average true distance modulus of $16.12 \pm 0.04$ or a distance of $16.7 \pm 0.3$ kpc. Comparison of the observational CMD with theoretical isochrones indicates an age of $12.5 \pm 2.0$ Gyrs for the cluster. A careful identification of all reported Blue Stragglers (BS) and their $V,I$ magnitudes leads to the conclusion that BS12, BS22, BS23 and BS24 are not BS. On the other hand, three new BS are reported. Variability was found in seven BS, very likely of the SX Phe type in five of them, and in one red giant star. The new SX Phe stars follow established $PL$ relationships and indicate a distance in agreement with the distance from the RR Lyrae stars.Comment: 21 pages, 16 figures, 11 tables. Accepted for publication in MNRA

PHE-SICH-CT-IDS: A Benchmark CT Image Dataset for Evaluation Semantic Segmentation, Object Detection and Radiomic Feature Extraction of Perihematomal Edema in Spontaneous Intracerebral Hemorrhage

Intracerebral hemorrhage is one of the diseases with the highest mortality and poorest prognosis worldwide. Spontaneous intracerebral hemorrhage (SICH) typically presents acutely, prompt and expedited radiological examination is crucial for diagnosis, localization, and quantification of the hemorrhage. Early detection and accurate segmentation of perihematomal edema (PHE) play a critical role in guiding appropriate clinical intervention and enhancing patient prognosis. However, the progress and assessment of computer-aided diagnostic methods for PHE segmentation and detection face challenges due to the scarcity of publicly accessible brain CT image datasets. This study establishes a publicly available CT dataset named PHE-SICH-CT-IDS for perihematomal edema in spontaneous intracerebral hemorrhage. The dataset comprises 120 brain CT scans and 7,022 CT images, along with corresponding medical information of the patients. To demonstrate its effectiveness, classical algorithms for semantic segmentation, object detection, and radiomic feature extraction are evaluated. The experimental results confirm the suitability of PHE-SICH-CT-IDS for assessing the performance of segmentation, detection and radiomic feature extraction methods. To the best of our knowledge, this is the first publicly available dataset for PHE in SICH, comprising various data formats suitable for applications across diverse medical scenarios. We believe that PHE-SICH-CT-IDS will allure researchers to explore novel algorithms, providing valuable support for clinicians and patients in the clinical setting. PHE-SICH-CT-IDS is freely published for non-commercial purpose at: https://figshare.com/articles/dataset/PHE-SICH-CT-IDS/23957937

Stable isotope containing peptides as probes of ligand—receptor interactions Deutero-formyl-methionyl-leucyl-phenylalanine

AbstractThe synthesis and biological evaluation of the stable isotope containing chemotactic peptide, deuteroformyl-methionyl-leucyl-phenylalanine (C2DO-Met-Leu-Phe-OH) is reported. The results, using lysosomal enzyme release from cytochalasin B-treated rabbit neutrophils for biological evaluation indicates a 3–4 fold enhancement over CHO-Met-Leu-Phe-OH itself. This is consistent with previous speculation that the formyl proton hydrogen bonds with a critical area of the chemotactic peptide receptor of rabbit neutrophils

Fidelity of Phenylalanyl-tRNA Synthetase in Binding the Natural Amino Acids

Aminoacyl-tRNA synthetases guard the fidelity of cognate amino acid incorporation during protein biosynthesis; for example, phenylalanyl-tRNA synthetase (PheRS) activates and transfers only Phe to its tRNA. Since we are interested in using a computational protocol to identify nonnatural amino acids that are incorporated by wild-type PheRS, it is critical to understand the fidelity of PheRS in binding the 20 natural amino acids. To this end, HierDock, a computational protocol for predicting binding sites and relative binding affinities, was used for testing the natural amino acids in PheRS. Scanning the entire ligand-accessible protein surface for the best binding region, we find that HierDock correctly identifies the active site of Phe in PheRS and predicts Phe within 0.61 Å RMSD of the crystal structure. HierDock also successfully shows PheRS discriminates for Phe, as the noncognate amino acids bind less favorably in the binding site of Phe. However, we find that Met, Cys, and Tyr bind competitively but at positions distant from the Phe binding site. This result corroborates in vitro measurements of aminoacyl adenylate formation, which show Met competes with Phe at the amino acid binding stage. We predict that the binding site of Met would not activate PheRS, as the noncognate amino acid cannot establish suitable hydrogen bonds with the PheRS reaction center. These results validate the use of HierDock in predicting the binding sites of the cognate amino acids in PheRS. The HierDock procedure calculates the discrimination of aminoacyl-tRNA synthetases at the stage of binding the cognate amino acid and offers a molecular level understanding of the mistakes made in protein biosynthesis that are not readily uncovered through experiments. This technique is also useful for predicting the binding of a selected nonnatural amino acid analogue, thereby indicating whether the molecule would be incorporated into a wild-type aminoacyl-tRNA synthetase

Numerical and Experimental Steady-State Investigation of Supercritical CO2 Gas Cooler Plate Heat Exchanger

Natural environmentally friendly refrigerants have been considered as alternatives to HFC refrigerants with high Global Warming Potential. In transcritical R744 vapor compression cycles, the heat rejection process occurs above the critical point where the temperature and the pressure are independent of each other. In this work, the gas cooling in a commercial 20 plate heat exchanger (PHE) used as gas cooler is modeled numerically and compared to experimental results. The numerical model is a simplified 2D model, i.e. corrugated plates with zero chevron angle. Four different real gas models are tested in terms of agreement and convergence, and numerical results from four cases with CO2 pressure ranging from 80 to 95 bar are compared with experimental results. Each case is numerically modeled with surface area that is set to match total surface area of the 20 PHE, and with one and half times the surface area of the 20 PHE. The increase in the surface area is shown to compensate for the chevron angle effect. For the experimental investigation, a CO2 heat pump test rig is used that supports cooling and heating applications. The numerical results show that the PHE outlet temperatures are in good agreement with the experimental results. The simplified 2D numerical models could reduce the computational costs associated with 3D PHE numerical simulations