Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography.

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

[Identification of Δ8-Tetrahydrocannabinol (THC) and Δ9-THC Analogs, with Different Lengths of Alkyl Chain at C-3 Position, in Oil Products Distributed on the Internet].

Since around 2021, products claiming to contain a Δ9-tetrahydrocannabinol (THC) analog with different lengths of alkyl chain at C-3 position have been sold on the internet in Japan. Δ9-THC has a pentyl group derived from the precursor olivetol at the C-3 position. These products include liquid cartridges for electronic cigarettes, herbal products, and gummy products. This study analyzed and determined the ingredients in five oil products distributed on the internet from 2022 to 2023 that claim to contain THC analogs. Samples of each product were used for GC-MS and LC-MS measurements. After isolating and purifying the unknown components from the products, structural analysis was performed by measuring 1H, 13C-NMR and various two-dimensional NMR [HH correlation spectroscopy (H-H COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY)]. The analysis identified Δ8-tetrahydrocannabivarin (THCV), Δ9-THCV, Δ8-tetrahydrocannabutol (THCB), Δ9-THCB, Δ8-tetrahydrocannabihexol (THCH), Δ9-THCH, Δ8-3-octyl-THC (THCjd) and Δ9-THCjd. These compounds were Δ8-THC or Δ9-THC analogs with different lengths of alkyl chain at C-3 position. Meanwhile, Δ4(8)-iso-THCV and Δ11-THCB were identified as minor components of the product, and were considered to be the reaction byproducts of the synthesis of the Δ8-THC or Δ9-THC analogs. In the future, there are concerns about the distribution of products containing new THC analogs. Therefore, continuous provision monitoring of newly detected in the products is important.

[Usefulness of web-based application for health surveys before and after peripheral blood stem cell harvest from healthy donors].

Health surveys to assess adverse events after peripheral blood stem cell harvest (PBSCH) have conventionally been conducted by phone, but phone calls are suboptimal for conducting frequent surveys. We developed a web-based application (donor app) that enables donors to inform healthcare professionals (HCPs) of their health status as an electronic patient-reported outcome (ePRO). In this prospective observational study, we compared the usefulness of this donor app to phone calls for conducting health surveys. App users reported ePRO daily, and patients called by HCPs reported their health status at least once a week when called. The observation period was from the first administration of granulocyte colony-stimulating factor to the first follow-up visit after PBSCH, excluding the hospitalization period. Each group consisted of eight donors with a median age of 32 years (range: 19-58). Nine (56.3%) were female. There were eight related donors in the phone call group and four in the donor app group. During the observation period, HCPs obtained health status reports more frequently from app users than from phone call recipients (mean proportion of days with reports made during the observation period, 27.0% vs 53.5%; p<0.05). Average time spent by the HCPs for one follow-up and total follow-ups were both significantly shorter when the donor app was used. There were no differences in donor burden or satisfaction with donation. Our study suggests that use of a donor app could provide more detailed health survey data without increasing the burden on donors and HCPs.

Estimation of threshold thickness of residual normal tissue in lung dysfunction detectable by dynamic chest radiography: A virtual imaging trial.

BACKGROUND: Dynamic chest radiography (DCR) is a recently developed functional x-ray imaging technique that detects pulmonary ventilation impairment as a decrease in changes in lung density during respiration. However, the diagnostic performance of DCR is uncertain owing to an insufficient number of clinical cases. One solution is virtual imaging trials (VITs), which is an emerging alternative method for efficiently evaluating medical imaging technology via computer simulation techniques. PURPOSE: This study aimed to estimate the typical threshold thickness of residual normal tissue below which the presence of emphysema may be detected by DCR via VITs using virtual patients with different physiques and a user-defined ground truth. METHODS: Twenty extended cardiac-torso (XCAT) phantoms that exhibited changes in lung density during respiration were generated to simulate virtual patients. To simulate a locally collapsed lung, an air sphere was inserted into each lung regions in the phantom. The XCAT phantom was virtually projected using an x-ray simulator. The respiratory changes in pixel value (ΔPV) were measured on the projected air spheres (simulated lesions) to calculate the percentage of decrease (ΔPV%) relative to ΔPVexp-ins in the absence of an air sphere. The relationship between the amount of residual normal tissue and ΔPV% was fitted to a cubic approximation curve (hereafter, performance curve), and the threshold at which the ΔPV% began to decrease (normal-tissuethre) was determined. The goodness of fit for each performance curve was evaluated according to the coefficient of determination (R2) and the 95% confidence interval derived from the standard errors between the measured and theoretical values corresponding to each performance curve. The ΔPV% was also visualized as a color scaling to validate the results of the VITs in both virtual and clinical patients. RESULTS: For each lung region in all body sizes, the ΔPV% decreased as the amount of residual normal tissue decreased and could be defined as a function of the amount of residual normal tissue in front of and behind the simulated lesions with high R2 values. Meanwhile, the difference between the measured and theoretical values corresponding to each performance curve was only partially included in the 95% confidence interval. The normal-tissuethre values were 146.0, 179.5, and 170.9 mm for the upper, middle, and lower lungs, respectively, which were demonstrated in virtual patients and one real patient, where the value of the residual normal tissue was less than that of normal-tissuethre; any reduction in the residual normal tissue was reflected as a reduced ΔPV and depicted as a reduced color intensity. CONCLUSIONS: The performance of DCR-based pulmonary impairment assessment depends on the amount of residual normal tissue in front of and behind the lesion rather than on the lesion size. The performance curve can be defined as a function of the amount of residual normal tissue in each lung region with a specific threshold of normal tissue remaining where lesions become detectable, shown as a decrease in ΔPV. The results of VITs are expected to accelerate future clinical trials for DCR-based pulmonary function assessment.

Proceedings Virtual Imaging Trials in Medicine 2024.

This submission comprises the proceedings of the 1st Virtual Imaging Trials in Medicine conference, organized by Duke University on April 22-24, 2024. The listed authors serve as the program directors for this conference. The VITM conference is a pioneering summit uniting experts from academia, industry and government in the fields of medical imaging and therapy to explore the transformative potential of in silico virtual trials and digital twins in revolutionizing healthcare. The proceedings are categorized by the respective days of the conference: Monday presentations, Tuesday presentations, Wednesday presentations, followed by the abstracts for the posters presented on Monday and Tuesday.

Association of placental weight at birth with maternal whole blood concentration of heavy metals (cadmium, lead, mercury, selenium, and manganese): The Japan Environment and Children's Study (JECS).

BACKGROUND: Lifelong health is dependent on prenatal growth and development, influenced by the placental intrauterine environment. Charged with dual functions--exchange of oxygen and nutrients as well as a barrier against toxins--the placenta itself is susceptible to environmental exposure to heavy metals. OBJECTIVE: To examine the use of placenta weight as a biomarker for heavy metal exposure using a large Japanese cohort of pregnant women. METHODS: The placenta weight, as a biomarker of exposure to heavy metals (cadmium, lead, and mercury), was investigated using data from the Japan Environment and Children's Study (2011-2014). Selenium and manganese were included as factors directly affecting fetal growth or heavy metal toxicity. Maternal blood samples collected in the second or third trimester were used to measure heavy metal concentrations. The association between maternal blood metal concentrations and placenta weight was explored by applying Z scores and multivariable logistic regression analysis and classifying participants into quartiles (Q1, Q2, Q3, and Q4) according to metal concentrations. RESULTS: This study included a total of 73,005 singleton pregnant women who delivered via live births and met the inclusion criteria. The median heavy metal concentrations in the maternal whole blood were 0.662 ng/g cadmium, 5.85 ng/g lead, 3.61 ng/g mercury, 168 ng/g selenium, and 15.3 ng/g manganese. Regression analysis revealed a significant correlation between placenta weight Z scores and maternal blood metal concentrations: cadmium, 0.0660 (standard error = 0.0074, p < 0.001); selenium, -0.3137 (standard error = 0.0276, p < 0.001); and manganese, 0.1483 (standard error = 0.0110, p < 0.001). CONCLUSION: This study provides a robust examination of the association between heavy metal exposure and placenta weight. Cadmium and manganese showed a positive correlation with significant differences, whereas selenium showed a negative correlation. Essential elements notably affect placenta weight differently. No significant association was noted between lead or mercury and placenta weight.

Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Identification of hexahydrocannabinol (HHC), dihydro-iso-tetrahydrocannabinol (dihydro-iso-THC) and hexahydrocannabiphorol (HHCP) in electronic cigarette cartridge products.

PURPOSE: Since 2021, products claiming to contain hexahydrocannabinol (HHC) and hexahydrocannabiphorol (HHCP), which are tetrahydrocannabinol (THC) analogs, have been distributed via the Internet. Owing to the presence of three asymmetric carbons in their structure, HHC and HHCP have multiple stereoisomers. This study aimed to identify the actual stereoisomers of HHC and HHCP isolated from electronic cigarette cartridge products using nuclear magnetic resonance (NMR) spectroscopy. METHODS: Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) were used for the analyses of two major peaks and one minor peak in product A and two major peaks in product B. These five compounds were isolated by silica gel column chromatography, and their structures were analyzed by 1H, 13C-NMR and various two-dimensional NMR techniques, i.e., H-H correlation spectroscopy, heteronuclear multiple quantum coherence, heteronuclear multiple-bond correlation, and nuclear Overhauser effect spectroscopy. RESULTS: Three compounds isolated from product A were identified as rel-(6aR,9R,10aR)-hexahydrocannabinol (11ß-hexahydrocannabinol; 11ß-HHC), rel-(6aR,9S,10aR)-hexahydrocannabinol (11α-hexahydrocannabinol, 11α-HHC), and a minor compound (2R,5S,6R)-dihydro-iso-tetrahydrocannabinol (dihydro-iso-THC). Meanwhile, the structural isomers of the major compound isolated from product B were identified as rel-(6aR, 9R, 10aR)-hexahydrocannabiphorol (11ß-hexahydrocannabiphorol; 11ß-HHCP) and rel-(6aR, 9S, 10aR)-hexahydrocannabiphorol (11α-hexahydrocannabiphorol; 11α-HHCP). CONCLUSIONS: The presence of both 11ß-HHC and 11α-HHC in the HHC products analyzed in this study suggests that they were most likely synthesized via the reduction reaction of Δ8-THC or Δ9-THC. Dihydro-iso-THC was probably obtained as a byproduct of the synthesis of Δ8-THC or Δ9-THC from cannabidiol. Similarly, 11ß-HHCP and 11α-HHCP in the HHCP product could stem from Δ9-tetrahydrocannabiphorol.

Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Deep learning-based cardiothoracic ratio measurement on chest radiograph: accuracy improvement without self-annotation.

Background: A reproducible and accurate automated approach to measuring cardiothoracic ratio on chest radiographs is warranted. This study aimed to develop a deep learning-based model for estimating the cardiothoracic ratio on chest radiographs without requiring self-annotation and to compare its results with those of manual measurements. Methods: The U-net architecture was designed to segment the right and left lungs and the cardiac shadow, from chest radiographs. The cardiothoracic ratio was then calculated using these labels by a mathematical algorithm. The initial model of deep learning-based cardiothoracic ratio measurement was developed using open-source 247 chest radiographs that had already been annotated. The advanced model was developed using a training dataset of 729 original chest radiographs, the labels of which were generated by the initial model and then screened. The cardiothoracic ratio of the two models was estimated in an independent test set of 120 original cases, and the results were compared to those obtained through manual measurement by four radiologists and the image-reading reports. Results: The means and standard deviations of the cardiothoracic ratio were 52.4% and 9.8% for the initial model, 51.0% and 9.3% for the advanced model, and 49.8% and 9.4% for the total of four manual measurements, respectively. The intraclass correlation coefficients (ICCs) of the cardiothoracic ratio ranged from 0.91 to 0.93 between the advanced model and the manual measurements, whereas those for the initial model and the manual measurements ranged from 0.77 to 0.82. Conclusions: Deep learning-based cardiothoracic ratio estimation on chest radiographs correlated favorably with the results obtained through manual measurements by radiologists. When the model was trained on additional local images generated by the initial model, the correlation with manual measurement improved even more than the initial model alone.

A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography.

Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.

Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography.

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

Association between maternal fish consumption during pregnancy and preterm births: the Japan Environment and Children's Study.

BACKGROUND: Fish are a rich source of essential nutrients that protect against preterm birth. However, as fish can absorb environmental pollutants, their consumption can also increase the risk of preterm birth. This study aimed to assess whether maternal fish consumption during pregnancy is associated with preterm birth in a nationwide large Japanese cohort that consumed relatively high amounts and many types of fish. METHODS: This study included 81,428 mother-child pairs enrolled in a nationwide prospective Japanese birth cohort study. Fish consumption was assessed using a validated food frequency questionnaire. Multivariate logistic regression was used to investigate the association of total consumption of fish, fatty fish and lean fish, fish paste, and seafood and clams with preterm birth, adjusted for potential confounders. RESULTS: There was no association between overall fish consumption and preterm births. However, the highest quintile of fish paste consumption was significantly associated with an increased risk of preterm birth (odds ratio [OR]: 1.11; 95% confidence interval [CI: 1.04, 1.17]). The consumption of baked fish paste at least three times per week was significantly associated with preterm birth (OR: 1.20; 95% CI: 1.03, 1.40). Consumption of other types of fish, except fish paste, was not significantly associated with preterm birth risk. CONCLUSIONS: Fish paste consumption may increase the risk of preterm birth. Further studies are required to confirm this association.

Characterization of the lysergic acid diethylamide analog, 1-(thiophene-2-carbonyl)-N,N-diethyllysergamide (1T-LSD) from a blotter product.

Recently, lysergic acid diethylamide (LSD) analogs have appeared worldwide as designer drugs. In this study, we identified a distributed LSD analog from a paper-sheet product. Gas chromatography-mass spectrometry (GC-MS), liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS), and liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were used to analyze the sheet product. The sheet product claimed to contain 1-(1,2-dimethylcyclobutanoyl)-N,N-diethyllysergamide (1D-LSD). However, an unknown compound was detected in the product together with tryptamine and L-tryptophan methyl ester. This compound was isolated from the sheets and identified as 1-(thiophene-2-carbonyl)-N,N-diethyl-6-methyl-9,10-didehydroergoline-8ß-carboxamide (1-thiophenoyl LSD; 1-(2-thienoyl)-LSD, 1T-LSD), using 1 H, 13 C nuclear magnetic resonance (NMR) spectroscopy and various two-dimensional NMR techniques. 1T-LSD was shown to have the thiophene-2-carbonyl group at the N1 position instead of the 1,2-dimethylcyclobutane-carbonyl group as claimed. The amount of 1T-LSD (free base) in three individual unit from one sheet was determined to be 87-100 µg per unit using a proton-specific quantitative NMR (1 H-qNMR) method. Deacylation of 1T-LSD to LSD was also observed to occur in methanol-d4 during NMR analysis. The UV spectrum of 1T-LSD differed from that of other LSD analogs, and the fluorescence sensitivity was much lower. Because of concerns about the future distribution of products containing new LSD analogs, continued monitoring of newly detected compounds in sheet products is encouraged.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump-probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ß-barrel across the time window of our measurements.