The reaction mechanism of the Ideonella sakaiensis PETase enzyme.

Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion.

Obtaining QM/MM binding free energies in the SAMPL8 drugs of abuse challenge: indirect approaches.

Accurately predicting free energy differences is essential in realizing the full potential of rational drug design. Unfortunately, high levels of accuracy often require computationally expensive QM/MM Hamiltonians. Fortuitously, the cost of employing QM/MM approaches in rigorous free energy simulation can be reduced through the use of the so-called "indirect" approach to QM/MM free energies, in which the need for QM/MM simulations is avoided via a QM/MM "correction" at the classical endpoints of interest. Herein, we focus on the computation of QM/MM binding free energies in the context of the SAMPL8 Drugs of Abuse host-guest challenge. Of the 5 QM/MM correction coupled with force-matching submissions, PM6-D3H4/MM ranked submission proved the best overall QM/MM entry, with an RMSE from experimental results of 2.43 kcal/mol (best in ranked submissions), a Pearson's correlation of 0.78 (second-best in ranked submissions), and a Kendall [Formula: see text] correlation of 0.52 (best in ranked submissions).

CIFDock: A novel CHARMM-based flexible receptor-flexible ligand docking protocol.

Docking studies play a critical role in the current workflow of drug discovery. However, limitations may often arise through factors including inadequate ligand sampling, a lack of protein flexibility, scoring function inadequacies (e.g., due to metals, co-factors, etc.), and difficulty in retaining explicit water molecules. Herein, we present a novel CHARMM-based induced fit docking (CIFDock) workflow that can circumvent these limitations by employing all-atom force fields coupled to enhanced sampling molecular dynamics procedures. Self-guided Langevin dynamics simulations are used to effectively sample relevant ligand conformations, side chain orientations, crystal water positions, and active site residue motion. Protein flexibility is further enhanced by dynamic sampling of side chain orientations using an expandable rotamer library. Steps in the procedure consisting of fixing individual components (e.g., the ligand) while sampling the other components (e.g., the residues in the active site of the protein) allow for the complex to adapt to conformational changes. Ultimately, all components of the complex-the protein, ligand, and waters-are sampled simultaneously and unrestrained with SGLD to capture any induced fit effects. This modular flexible docking procedure is automated using CHARMM scripting, interfaced with SLURM array processing, and parallelized to use the desired number of processors. We validated the CIFDock procedure by performing cross-docking studies using a data set comprised of 21 pharmaceutically relevant proteins. Five variants of the CHARMM-based SWISSDOCK scoring functions were created to quantify the results of the final generated poses. Results obtained were comparable to, or in some cases improved upon, commercial docking program data.

Purcell-Enhanced Spontaneous Emission of Molecular Vibrations.

Infrared (IR) spectroscopy of molecular vibrations provides insight into molecular structure, coupling, and dynamics. However, picosecond scale intermolecular and intramolecular many-body interactions, nonradiative relaxation, absorption, and thermalization typically dominate over IR spontaneous emission. We demonstrate how coupling to a resonant IR antenna can enhance spontaneous emission of molecular vibrations. Using time-domain nanoprobe spectroscopy we observe an up to 50% decrease in vibrational dephasing time T_{2,vib}, based on the coupling-induced population decay with T_{κ}≃550 fs and an associated Purcell factor of >10^{6}. This rate enhancement of the spontaneous emission of antenna-coupled molecular vibrations opens new avenues for IR coherent control, quantum information processing, and quantum chemistry.

Imaging Carrier Inhomogeneities in Ambipolar Tellurene Field Effect Transistors.

The development of van der Waals (vdW) homojunction devices requires materials with narrow bandgaps and simultaneously high hole and electron mobilities for bipolar transport, as well as methods to image and study spatial variations in carrier type and associated conductivity with nanometer spatial resolution. Here, we demonstrate the general capability of near-field scanning microwave microscopy (SMM) to image and study the local carrier type and associated conductivity in operando by studying ambiploar field-effect transistors (FETs) of the 1D vdW material tellurium in 2D form. To quantitatively understand electronic variations across the device, we produce nanometer-resolved maps of the local carrier equivalence backgate voltage. We show that the global device conductivity minimum determined from transport measurements does not arise from uniform carrier neutrality but rather from the continued coexistence of p-type regions at the device edge and n-type regions in the interior of our micrometer-scale devices. This work both underscores and addresses the need to image and understand spatial variations in the electronic properties of nanoscale devices.

Characterization and engineering of a plastic-degrading aromatic polyesterase.

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/ß-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

The ball in play demands of international rugby union.

OBJECTIVES: Rugby union is a high intensity intermittent sport, typically analysed via set time periods or rolling average methods. This study reports the demands of international rugby union via global positioning system (GPS) metrics expressed as mean ball in play (BiP), maximum BiP (max BiP), and whole match outputs. DESIGN: Single cohort cross sectional study involving 22 international players, categorised as forwards and backs. METHODS: A total of 88 GPS files from eight international test matches were collected during 2016. An Opta sportscode timeline was integrated into the GPS software to split the data into BiP periods. Metres per min (mmin-1), high metabolic load per min (HML), accelerations per min (Acc), high speed running per min (HSR), and collisions per min (Coll) were expressed relative to BiP periods and over the whole match (>60min). RESULTS: Whole match metrics were significantly lower than all BiP metrics (p<0.001). Mean and max BiP HML, (p<0.01) and HSR (p<0.05) were significantly higher for backs versus forwards, whereas Coll were significantly higher for forwards (p<0.001). In plays lasting 61s or greater, max BiP mmin-1 were higher for backs. Max BiP mmin-1, HML, HSR and Coll were all time dependant (p<0.05) showing that both movement metrics and collision demands differ as length of play continues. CONCLUSIONS: This study uses a novel method of accurately assessing the BiP demands of rugby union. It also reports typical and maximal demands of international rugby union that can be used by practitioners and scientists to target training of worst-case scenario's equivalent to international intensity. Backs covered greater distances at higher speeds and demonstrated higher HML, in general play as well as 'worst case scenarios'; conversely forwards perform a higher number of collisions.

Nanoimaging and Control of Molecular Vibrations through Electromagnetically Induced Scattering Reaching the Strong Coupling Regime.

Optical resonators can enhance light-matter interaction, modify intrinsic molecular properties such as radiative emission rates, and create new molecule-photon hybrid quantum states. To date, corresponding implementations are based on electronic transitions in the visible spectral region with large transition dipoles yet hampered by fast femtosecond electronic dephasing. In contrast, coupling molecular vibrations with their weaker dipoles to infrared optical resonators has been less explored, despite long-lived coherences with 2 orders of magnitude longer dephasing times. Here, we achieve excitation of molecular vibrations through configurable optical interactions of a nanotip with an infrared resonant nanowire that supports tunable bright and nonradiative dark modes. The resulting antenna-vibrational coupling up to 47 ± 5 cm-1 exceeds the intrinsic dephasing rate of the molecular vibration, leading to hybridization and mode splitting. We observe nanotip-induced quantum interference of vibrational excitation pathways in spectroscopic nanoimaging, which we model classically as plasmonic electromagnetically induced scattering as the phase-controlled extension of the classical analogue of electromagnetically induced transparency and absorption. Our results present a new regime of IR spectroscopy for applications of vibrational coherence from quantum computing to optical control of chemical reactions.

Infrared vibrational nanocrystallography and nanoimaging.

Molecular solids and polymers can form low-symmetry crystal structures that exhibit anisotropic electron and ion mobility in engineered devices or biological systems. The distribution of molecular orientation and disorder then controls the macroscopic material response, yet it is difficult to image with conventional techniques on the nanoscale. We demonstrated a new form of optical nanocrystallography that combines scattering-type scanning near-field optical microscopy with both optical antenna and tip-selective infrared vibrational spectroscopy. From the symmetry-selective probing of molecular bond orientation with nanometer spatial resolution, we determined crystalline phases and orientation in aggregates and films of the organic electronic material perylenetetracarboxylic dianhydride. Mapping disorder within and between individual nanoscale domains, the correlative hybrid imaging of nanoscale heterogeneity provides insight into defect formation and propagation during growth in functional molecular solids.

Single-step sub-200 fs mid-infrared generation from an optical parametric oscillator synchronously pumped by an erbium fiber laser.

We demonstrate the single-step generation of mid-infrared femtosecond laser pulses in a AgGaSe2 optical parametric oscillator that is synchronously pumped by a 100 MHz repetition rate sub-90 fs erbium fiber laser. The tuning range of the idler beam in principle covers ∼3.5 to 17 µm, only dependent on the choice of cavity and mirror design. As an example, we experimentally demonstrate idler pulse generation from 4.8 to 6.0 µm optimized for selective vibrational resonant molecular spectroscopy. We find an oscillation threshold as low as 150 mW of pump power. At 300 mW pump power and a central wavelength of ∼5.0 µm, we achieve an average infrared power of up to 17.5 mW, with a photon conversion efficiency of ∼18%. A pulse duration of ∼180 fs is determined from a nonlinear cross-correlation with residual pump light. The single-step nonlinear conversion leads to a high power stability with <1% average power drift at <0.5% rms noise over 1 h.

Correlative infrared nanospectroscopic and nanomechanical imaging of block copolymer microdomains.

Intermolecular interactions and nanoscale phase separation govern the properties of many molecular soft-matter systems. Here, we combine infrared vibrational scattering scanning near-field optical microscopy (IR s-SNOM) with force-distance spectroscopy for simultaneous characterization of both nanoscale optical and nanomechanical molecular properties through hybrid imaging. The resulting multichannel images and correlative analysis of chemical composition, spectral IR line shape, modulus, adhesion, deformation, and dissipation acquired for a thin film of a nanophase separated block copolymer (PS-b-PMMA) reveal complex structural variations, in particular at domain interfaces, not resolved in any individual signal channel alone. These variations suggest that regions of multicomponent chemical composition, such as the interfacial mixing regions between microdomains, are correlated with high spatial heterogeneity in nanoscale material properties.

Infrared Vibrational Nanospectroscopy by Self-Referenced Interferometry.

Infrared vibrational scattering scanning near-field optical microscopy (s-SNOM) has emerged as a new frontier in imaging science due to its potential to provide nanoscale spatially resolved chemical spectroscopy for the investigation of molecular, soft-matter, and biological materials. As a phase-sensitive technique able to yield the full complex dielectric function of materials, different interferometric schemes have been developed involving asymmetric interferometry between sample and reference arms. In this work, we take advantage of a greatly simplified symmetric geometry that uses the spatially coherent background scattered light from within the confocal sample volume as a reference field for signal amplification in both self-homodyne and self-heterodyne interferometry. On the basis of a simple model for tip-sample scattering and interferometric detection, we demonstrate the measurement of the vibrational response of molecular materials in good agreement with established values. In addition to a compact design, enhanced signal levels, and a reduced sensitivity to fluctuations and drift, including those from the light source, self-referenced interferometry brings benefits for routine s-SNOM chemical spectroscopy, remaining robust even under a wide range of challenging experimental environments.

Infrared Chemical Nano-Imaging: Accessing Structure, Coupling, and Dynamics on Molecular Length Scales.

This Perspective highlights recent advances in infrared vibrational chemical nano-imaging. In its implementations of scattering scanning near-field optical microscopy (s-SNOM) and photothermal-induced resonance (PTIR), IR nanospectroscopy provides few-nanometer spatial resolution for the investigation of polymer, biomaterial, and related soft-matter surfaces and nanostructures. Broad-band IR s-SNOM with coherent laser and synchrotron sources allows for chemical recognition with small-ensemble sensitivity and the potential for sensitivity reaching the single-molecule limit. Probing selected vibrational marker resonances, it gives access to nanoscale chemical imaging of composition, domain morphologies, order/disorder, molecular orientation, or crystallographic phases. Local intra- and intermolecular coupling can be measured through frequency shifts of a vibrational marker in heterogeneous environments and associated inhomogeneities in vibrational dephasing. In combination with ultrafast spectroscopy, the vibrational coherent evolution of homogeneous sub-ensembles coupled to their environment can be observed. Outstanding challenges are discussed in terms of extensions to coherent and multidimensional spectroscopies, implementation in liquid and in situ environments, general sample limitations, and engineering s-SNOM scanning probes to better control the nano-localized optical excitation and to increase sensitivity.

Vibrational nano-spectroscopic imaging correlating structure with intermolecular coupling and dynamics.

Molecular self-assembly, the function of biomembranes and the performance of organic solar cells rely on nanoscale molecular interactions. Understanding and control of such materials have been impeded by difficulties in imaging their properties with the desired nanometre spatial resolution, attomolar sensitivity and intermolecular spectroscopic specificity. Here we implement vibrational scattering-scanning near-field optical microscopy with high spectral precision to investigate the structure-function relationship in nano-phase separated block copolymers. A vibrational resonance is used as a sensitive reporter of the local chemical environment and we image, with few nanometre spatial resolution and 0.2 cm(-1) spectral precision, solvatochromic Stark shifts and line broadening correlated with molecular-scale morphologies. We discriminate local variations in electric fields between nano-domains with quantitative agreement with dielectric continuum models. This ability to directly resolve nanoscale morphology and associated intermolecular interactions can form a basis for the systematic control of functionality in multicomponent soft matter systems.

The effects of ambient lighting in chest radiology reading rooms.

Under typical dark chest radiography reading room conditions, a radiologist's pupils contract and dilate as their visual focus intermittently shifts between the high luminance monitor and the darker background wall, resulting in increased visual fatigue and degradation of diagnostic performance. A controlled increase of ambient lighting may minimize these visual adjustments and potentially improve comfort and accuracy. This study was designed to determine the effect of a controlled increase of ambient lighting on chest radiologist nodule detection performance. Four chest radiologists read 100 radiographs (50 normal and 50 containing a subtle nodule) under low (E=1 lx) and elevated (E=50 lx) ambient lighting levels on a DICOM-calibrated, medical-grade liquid crystal display. Radiologists were asked to identify nodule locations and rate their detection confidence. A receiver operating characteristic (ROC) analysis of radiologist results was performed and area under ROC curve (AUC) values calculated for each ambient lighting level. Additionally, radiologist selection times under both illuminance conditions were determined. Average AUC values did not significantly differ (p>0.05) between ambient lighting levels (estimated mean difference=-0.03; 95% CI, (-0.08, 0.03)). Average selection times decreased or remained constant with increased illuminance. The most considerable decreases occurred for false positive identification times (35.4±18.8 to 26.2±14.9 s) and true positive identification times (29.7±18.3 to 24.5±15.5 s). No performance differences were statistically significant. Study findings suggest that a controlled increase of ambient lighting within darkly lit chest radiology reading rooms, to a level more suitable for performance of common radiological tasks, does not appear to have a statistically significant effect on nodule detection performance.

The influence of increased ambient lighting on mass detection in mammograms.

RATIONALE AND OBJECTIVES: Recent research has provided evidence that in reading rooms equipped with liquid crystal displays (LCDs), a measured increase of ambient lighting may improve clinicians' detection performance. In agreement with this research, the American College of Radiology (ACR) has recommended a moderate increase of ambient lighting in mammography reading rooms. This study was designed to examine the effect of a controlled increase of ambient lighting in mammography reading rooms on the diagnostic performance of breast imaging radiologists. MATERIALS AND METHODS: Four breast imaging radiologists read 86 mammograms (43 containing subtle cancerous masses and 43 normal) under low (E = 1 lux) and elevated (E = 50 lux) ambient lighting levels on a Digital Imaging and Communications in Medicine-calibrated, medical-grade LCD. Radiologists were asked to identify cancerous masses and to rate their detection confidence. Observer areas under the curve (AUCs) were calculated using a receiver-operating characteristic analysis of fully paired results. Additionally, average observer selection times under both ambient lighting levels were determined. RESULTS: Average radiologist AUCs decreased with elevated ambient lighting (0.78 +/- 0.03 to 0.72 +/- 0.04). Observer performance differences, however, were of the same order of magnitude as interobserver variability and were not statistically significant. Average selection times under increased ambient lighting remained constant or decreased, with the greatest decrease occurring for false-positive (20.4 +/- 18.9 to 14.4 +/- 9.6 seconds) and true-positive (18.0 +/- 13.8 to 12.9 +/- 9.4 seconds) selections. CONCLUSION: The results agree with those of previous studies in that observer performance differences under a controlled increase of ambient lighting are not statistically significant. On the basis of these findings and ACR guidelines, a moderate increase of ambient lighting in mammography reading rooms is still suggested, but further research with additional cases and observers should be considered.

Object detectability at increased ambient lighting conditions.

Under typical dark conditions encountered in diagnostic reading rooms, a reader's pupils will contract and dilate as the visual focus intermittently shifts between the high luminance display and the darker background wall, resulting in increased visual fatigue and the degradation of diagnostic performance. A controlled increase of ambient lighting may, however, reduce the severity of these pupillary adjustments by minimizing the difference between the luminance level to which the eyes adapt while viewing an image (L(adp)) and the luminance level of diffusely reflected light from the area surrounding the display (L(s)). Although ambient lighting in reading rooms has conventionally been kept at a minimum to maintain the perceived contrast of film images, proper Digital Imaging and Communications in Medicine (DICOM) calibration of modern medical-grade liquid crystal displays can compensate for minor lighting increases with very little loss of image contrast. This paper describes two psychophysical studies developed to evaluate and refine optimum reading room ambient lighting conditions through the use of observational tasks intended to simulate real clinical practices. The first study utilized the biologic contrast response of the human visual system to determine a range of representative L(adp) values for typical medical images. Readers identified low contrast horizontal objects in circular foregrounds of uniform luminance (5, 12, 20, and 30 cd/m2) embedded within digitized mammograms. The second study examined the effect of increased ambient lighting on the detection of subtle objects embedded in circular foregrounds of uniform luminance (5, 12, and 35 cd/m2) centered within a constant background of 12 cd/m2 luminance. The images were displayed under a dark room condition (1 lux) and an increased ambient lighting level (50 lux) such that the luminance level of the diffusely reflected light from the background wall was approximately equal to the image L(adp) value of 12 cd/m2. Results from the first study demonstrated that observer true positive and false positive detection rates and true positive detection times were considerably better while viewing foregrounds at 12 and 20 cd/m2 than at the other foreground luminance levels. Results from the second study revealed that under increased room illuminance, the average true positive detection rate improved a statistically significant amount from 39.3% to 55.6% at 5 cd/m2 foreground luminance. Additionally, the true positive rate increased from 46.4% to 56.6% at 35 cd/m2 foreground luminance, and decreased slightly from 90.2% to 87.5% at 12 cd/m2 foreground luminance. False positive rates at all foreground luminance levels remained approximately constant with increased ambient lighting. Furthermore, under increased room illuminance, true positive detection times declined at every foreground luminance level, with the most considerable decrease (approximately 500 ms) at the 5 cd/m2 foreground luminance. The first study suggests that L(adp) of typical mammograms lies between 12 and 20 cd/m2, leading to an optimum reading room illuminance of approximately 50-80 lux. Findings from the second study provide psychophysical evidence that ambient lighting may be increased to a level within this range, potentially improving radiologist comfort, without deleterious effects on diagnostic performance.