Substrates reconfigure picosecond electric field fluctuations in dihydrofolate reductase

Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.

Validation of two calculation options built in Elekta Monaco Monte Carlo based algorithm using MCNP code

The migration characteristics of open-end and sheath fibres of core-sheath friction spun yarn have been studied using the tracer fibre technique. In open-end friction spun yarns, migration parameters were studied in both two-dimensional and three-dimensional projection methods. From the analysis, it has been observed that three-dimensional studies give higher mean fibre position (MFP), root mean square (RMS) deviation and mean migration intensity (MMI) than two-dimensional studies. The results show that fibres from first sliver feeding position have significantly higher MFP, RMS deviation and MMI than fifth sliver feeding position. The results also show that trailing half of fibres for first sliver feeding position has higher MFP, RMS deviation and MMI than leading half of fibres for both types of yarns. The migration parameters are mainly influenced by suction air pressure followed by opening roller speed and difference in drum speed for both open-end and core-sheath friction yarns. In open-end friction yarns for first and fifth sliver feeding positions, the experimental variables show different trends for MFP and RMS deviation.

ABSTRACTObjectiveThis study aimed to investigate the accuracy of conventional and digital impression‐making and cast‐fabrication using coded healing abutments on an edentulous mandibular model under in vitro conditions.Materials and MethodsOur study investigated the accuracy of the On1 Concept (Nobel Biocare; Kloten, Switzerland) coded healing abutment system using conventional and digital workflows. The Conical Connection (CC) system (Nobel Biocare; Kloten, Switzerland) was used as the control group in both workflows. 10–10 open‐tray impressions and intraoral scans were made from the reference model with each system. Models built from intraoral scans were additively fabricated, and open‐tray impressions were poured with type‐4 dental stone. The prepared models were digitized using a desktop scanner with an accuracy of 4 μm (E4, 3Shape; Copenhagen, Denmark) and superimposed on the reference scan. Four linear distances and root mean square (RMS) deviations were measured using metrology software.ResultsFive dimensions were measured using signed and absolute deviations, resulting in nine outcomes. RMS and diagonal deviations provided the most insight into overall model deviations. Mean RMS deviations were: 58.30 (14.95) μm for CC_conv, 47.66 (13.04) μm for On1_conv, 204.97 (37.40) μm for CC_dig, and 136.64 (13.49) μm for On1_dig. Significant differences were found between On1_conv vs. CC_dig, On1_conv vs. On1_dig, and CC_conv vs. CC_dig. Mean linear deviations between the molar positions were: 24.49 (58.20) μm for CC_conv, 87.46 (106.70) μm for On1_conv, −104.76 (125.83) μm for CC_dig, and 140.64 (190.56) μm for On1_dig. Significant differences were observed between On1_conv vs. CC_dig and CC_dig vs. On1_dig.ConclusionsBased on the RMS deviations, the conventional method is significantly more accurate at both implant and platform levels in the case of an in vitro edentulous lower jaw model. The RMS deviations of the implant analogs are smaller on the platform level with both conventional and digital methods.

The discovery of antibiotics initiated the era of drug innovation and implementation for human and animal health. Very soon, antibiotic resistance started evolving due to over-prescription and heavy usage of drugs leading to deleterious side effects. However, using plant extracts or medicinal plants has emerged as a new approach to dealing with the current problem. One such medicinal plant Sisymbrium irio L. is widely used in Unani therapy as an antimicrobial, analgesic, antipyretic, antioxidant, anti-inflammatory, hepatoprotective, bronchoprotective etc. The phytochemicals extracted from the aerial part of the plant have been used as a natural compound library and screened against a well-known anti-bacterial drug target Dihydrofolate reductase (DHFR) enzyme of Staphylococcus aureus. The top two phytochemicals with lower docking score along with the positive control were subjected to molecular dynamics (MD) simulation studies to examine the stabilities of the complexes over 100 ns, followed by binding free energy estimation. The Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) and Radius of Gyration (Rg) yielded established results throughout the MD run. Moreover, the derived phytochemicals exhibited lower binding free energy values than the positive control that can be tested for its in vitro efficacy, followed by further optimization to attain a potent therapeutic against S. aureus. Taken together, the present study suggests two promising phytochemicals derived from the aerial part of the plant S. irio with stable MD simulation results, strong binding affinity and no side effects.

Abstract. A new microwave satellite water vapour retrieval for the polar winter atmosphere is presented. The retrieval builds on the work of Miao et al. (2001) and Melsheimer and Heygster (2008), employing auxiliary information for atmospheric conditions and numerical optimization. It was tested using simulated and actual measurements from the Microwave Humidity Sounder (MHS) satellite instruments. Ground truth was provided by the G-band vapour radiometer (GVR) at Barrow, Alaska. For water vapour columns less than 6 kg m−2, comparisons between the retrieval and GVR result in a root mean square (RMS) deviation of 0.39 kg m−2 and a systematic bias of 0.08 kg m−2. These results are compared with RMS deviations and biases at Barrow for the retrieval of Melsheimer and Heygster (2008), the AIRS and MIRS satellite data products, and the ERA-Interim, NCEP, JRA-55, and ASR reanalyses. When applied to MHS measurements, the new retrieval produces a smaller RMS deviation and bias than for the earlier retrieval and satellite data products. The RMS deviations for the new retrieval were comparable to those for the ERA-Interim, JRA-55, and ASR reanalyses; however, the MHS retrievals have much finer horizontal resolution (15 km at nadir) and reveal more structure. The new retrieval can be used to obtain pan-Arctic maps of water vapour columns of unprecedented quality. It may also be applied to measurements from the Special Sensor Microwave/Temperature 2 (SSM/T2), Advanced Microwave Sounding Unit B (AMSU-B), Special Sensor Microwave Imager/Sounder (SSMIS), Advanced Technology Microwave Sounder (ATMS), and Chinese MicroWave Humidity Sounder (MWHS) instruments.

The New York Structural GenomiX Research Consortium (NYSGXRC) has selected the protein coded by yxaF gene from Bacillus subtilis as a target for structure determination. The yxaF protein has 191 residues with a molecular mass of 21 kDa and had no sequence homology to any structure in the Protein Data Bank (PDB) at the time of target selection. We aimed to elucidate the three-dimensional structure for the putative protein yxaF to better understand the relationship between protein sequence, structure, and function. This protein is annotated as a putative helix-turn-helix (HTH) type transcriptional regulator. Many transcriptional regulators like TetR and QacR use a structurally well-defined DNA-binding HTH motif to recognize the target DNA sequences. DNA-HTH motif interactions have been extensively studied. As the HTH motif is structurally conserved in many regulatory proteins, these DNA-protein complexes show some similarity in DNA recognition patterns. Many such regulatory proteins have a ligand-binding domain in addition to the DNA-binding domain. Structural studies on ligand-binding regulatory proteins provide a wealth of information on ligand-, and possibly drug-, binding mechanisms. Understanding the ligand-binding mechanism may help overcome problems with drug resistance, which represent increasing challenges in medicine. The protein encoded by yxaF, hereafter called T1414, shows foldmore » similar to QacR repressor and TetR/CamR repressor and possesses putative DNA and ligand-binding domains. Here, we report the crystal structure of T1414 and compare it with structurally similar drug and DNA-binding proteins.« less

Fingerprinting DHFR in Single-Molecule AFM Studies

An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

Fungal infection of invasive nature is an alarming threat globally and a leading cause of human morbidity and mortality as they are opportunistic in nature. Rising resistance to current clinically approved marketed products for fungal infections is a major concern for humans. Dihydrofolate Reductase (DHFRase) is an essential enzyme in folate metabolic pathway responsible for DNA synthesis and is ubiquitous to all organisms, and also acts as a key target for developing antifungal drugs. In this study, potential mutant DHFRase inhibitors were screened with the help of hierarchical mode of docking of virtual library of antifungal compounds and molecular dynamic (MD) simulation. The identification of best hits was done by using the docking, binding energy prediction and further, which was supported by their predicted pharmacokinetics. MD simulation of the human DHFRase enzyme with the reference lead compound i.e. PY957 and most promising hit found i.e. ChemDiv-C390-0455 and to validate the stability of enzyme-ligand complex in best 07 retrieved hit as a potential mutant DHFRase inhibitor. The key residues Glh30, Phe34, Phe64, Phe31 of the binding pocket acknowledged as essential were found to be matching with the key interactions of the selected hit. Computed root mean square deviation (RMSD) and root mean square fluctuation (RMSF) in MD simulation of complex of DHFRase enzyme with PY957 and ChemDiv-C390-0455 were read less than 2.25[Formula: see text]Å during 100 nanoseconds simulation for both complex.

Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.

Understanding set-up uncertainty effects on dose distributions is an important clinical problem but difficult to model accurately due to their dependence on tissue inhomogeneities and changes in the surface contour (i.e., variant effects). The aims are: (1) to evaluate and quantify the invariant and variant effects of set-up uncertainties, contour changes and tissue inhomogeneities on target dose-volume histograms (DVHs); (2) to propose a method to interpolate (variant) DVHs. We present a lung cancer patient to estimate the significance of set-up uncertainties, contour changes and tissue inhomogeneities in target DVHs. Differential DVHs are calculated for 15 displacement errors (with respect to the isocenter) using (1) an invariant shift of the dose distribution at the isocenter, (2) a full variant calculation, and (3) a B-spline interpolation applied to sparsely sampled variant DVHs. The collapsed cone algorithm was used for all dose calculations. Dosimetric differences are quantified with the root mean square (RMS) deviation and the equivalent uniform dose (EUD). To determine set-up uncertainty effects, weighted mean EUDs, assuming normally distributed displacement errors, are used. The maximum absolute difference and RMS deviation in the integral DVHs' relative dose between (1) the invariant and calculated curves are 65.2% and 5.8% and (2) the interpolated and calculated curves are 16.9% and 2.5%. Similarly, the maximum absolute difference and RMS deviation in mean EUD as a function of the set-up uncertainty's standard deviation between (1) the invariant and calculated curves are 0.02 and 0.01 Gy; and (2) the interpolated and calculated curves are 0.01 and 0.006 Gy. Since a "worst-case" example is selected, we conclude that, in the majority of clinical cases, the variant effects of contour changes, tissue inhomogeneities and set-up uncertainties on EUD are negligible. Interpolation is a valid, efficient method to approximate DVHs.

Trueness of complete arch intraoral implant scans using splinted and non-splinted scan bodies: A systematic review and meta-analysis.

Any PhD student or postdoc, who scrambles to write and submit a publication before getting scooped, can testify that science is a fast‐moving endeavor. Given our limited time and the ever‐increasing pace with which scientific studies are published, few students and postdocs—and PIs as a matter of fact—have time to keep up with the literature. “Reading” a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. Notwithstanding these constraints, I would argue that the scientific literature is a treasure trove of information and ideas that go beyond contemporary papers to include classical publications. However, it can be difficult to motivate students and postdocs to read old landmark publications that reported groundbreaking discoveries and opened up new avenues of research. Many think that these papers are of merely historic interest and have little to contribute to scientists expected to use cutting‐edge methods to produce high‐impact publications. > Reading a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. However, classic papers still have a lot to offer. For once, they withstood the test of the time, as the reported results have shown to be correct and reproducible. This is unfortunately not the case for many high‐impact publications today, which, owing to the hype of selling it to the highest impact factor journal, just tell a cool story that in reality may be much less clear‐cut than reported. Furthermore, classic scientific publications often impress by a conceptual clarity and in many cases simplicity and thereby offer valuable lessons of how best science should be practiced. These are qualities I miss in many of today's papers that contain exceedingly large data sets …