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.

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.

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.

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.

The effect of mask fit test on the association between the concentration of metals in biological samples and the results of time-weighted average personal exposure: A study on Japanese male welders.

OBJECTIVES: The mask fit test confirms whether the wearing condition of the wearer's face and the facepiece of the respirators are used appropriately. This study aimed to examine whether the results of the mask fit test affect the association between the concentration of metals related to welding fumes in biological samples and the results of time-weighted average (TWA) personal exposures. METHODS: A total of 94 male welders were recruited. Blood and urine samples were obtained from all participants to measure the metal exposure levels. Using personal exposure measurements, the 8-h TWA (8 h-TWA) of respirable dust, TWA of respirable Mn, and 8-h TWA of respirable Mn were calculated. The mask fit test was performed using the quantitative method specified in the Japanese Industrial Standard T8150:2021. RESULTS: Fifty-four participants (57%) passed the mask fit test. Only in the Fail group of the mask fit test, it was observed that blood Mn concentrations be positively associated with the results of TWA personal exposure after adjusting for multivariate factors (8-h TWA of respirable dust; coefficient, 0.066; standard error (SE), 0.028; P = 0.018, TWA of respirable Mn: coefficient, 0.048; SE, 0.020; P = 0.019, 8 h-TWA of respirable Mn: coefficient, 0.041; SE, 0.020; P = 0.041). CONCLUSIONS: The results clarify that welders with high concentrations of welding fumes in their breathing air zone are exposed to dust and Mn if there is leaking air owing to the lack of fitness between respirators and the wearer's face when using human samples in Japan.

Preoperative assessment of pleural adhesions in patients with lung cancer based on quantitative motion analysis with dynamic chest radiography: A retrospective study.

PURPOSE: Preoperative assessment of pleural adhesion is crucial for appropriate surgical planning. This study aimed to quantitatively evaluate the usefulness of motion analysis using dynamic chest radiography (DCR) for assessing pleural adhesions. METHODS: Sequential chest radiographs of 146 lung cancer patients with or without pleural adhesions (n = 25/121) were obtained using a DCR system during respiration (registration number: 1729). The local motion vector was measured, and the percentage of poor motion area to the maximum expiration lung area (%lung area with poor motion) was calculated. Subsequently, percentage values ≥49.0% were considered to indicate pleural adhesions. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction performance. The percentage of lung area with poor motion was compared between patients with and without pleural adhesions (p < 0.05). RESULTS: DCR-based motion analysis correctly predicted pleural adhesions in 21 out of 25 patients, with 47 false-positive results (sensitivity, 84.0%; specificity, 61.2%; PPV, 30.9%; NPV, 94.9%). The lung with pleural adhesions showed a significantly greater %lung area with poor motion than the opposite lung in the same patient, similar to the cancerous lung in patients without pleural adhesions. CONCLUSION: On DCR-based motion analysis, pleural adhesions could be indicated by an increase in the percentage of lung area with poor motion. Although the proposed method cannot identify the exact location of pleural adhesions, information regarding the presence or absence of pleural adhesions provided by DCR would help surgeons prepare for challenging surgeries and obtain informed consent from patients.

Nudge for Environmental Restructuring in Diabetes Self-Management: Comment on Existing Systematic Reviews.

Diabetes self-management education and support are necessary for all people living with diabetes, but its accessibility is limited worldwide. Nudge strategies have been proposed as an environmental outreach for diabetes management. This article provides further insights regarding environmental restructuring nudges into the cumulative evidence on diabetes self-management interventions from existing systematic reviews that classified primary trials using the behavior change technique taxonomy (BCTTv1). Among the 137 relevant articles searched through the bibliographic databases until 2022, three systematic reviews were scrutinized. Environmental restructuring nudges have been tested in interpersonal communications for diabetes self-management. Although nudge-based techniques were used with other types of behavior techniques in various trial contexts, the independent effects of social restructuring nudges were not denied in previous meta-analyses. Environmental restructuring nudges may be feasible in diabetes management, but they are still controversial with internal and external validation. Considering care accessibility for diabetes management, social restructuring nudges applied to healthcare providers are expected to complement healthcare systems. For future implementation, the rationale for the practice should be explicit in the conceptualization and evidence synthesis of diabetes-specific nudge interventions based on global sources.

[Determination of 11 Cannabinoids in Cannabis sativa L. by Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-TOF-MS)].

Eleven major cannabinoids from each subdivided tissue of drug-type and fiber-type cannabis plants were determined by means of a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). The cannabinoids analyzed in this study were tetrahydrocannabinol acid (THCA), Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol acid (CBDA), cannabidiol (CBD), Δ8-tetrahydrocannabinol (Δ8-THC), cannabinol (CBN), cannabichromene (CBC), cannabidivarin (CBDV), cannabigerolic acid (CBGA), cannabigerol (CBG) and tetrahydrocannabivarin (THCV). As a result, THCA was detected in the bracts at 28.4 µg/mg, in the buds at 24.8 µg/mg, and in the leaves at 5.1 to 10.5 µg/mg in the drug-type cannabis plant. In addition, Δ9-THC, CBGA, CBN, CBG, CBC, and THCV were mainly detected in bracts, buds, and leaves. On the other hand, as for the fiber-type cannabis plant, CBDA was detected in the bracts at 27.5 µg/mg, in the buds at 10.6 µg/mg, and in the leaves at 1.5-3.3 µg/mg. In addition, Δ9-THCA, CBD, Δ9-THC, CBC, and CBG were mainly detected in bracts, buds, and leaves.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Association between maternal socioeconomic status and breastfeeding: Results from the Japan environment and children's study.

Although breastfeeding has various benefits for mothers and children, there are several barriers to continuing breastfeeding practices. However, little is known about the relationship between breastfeeding in Japan and maternal socioeconomic circumstances. Based on data from the Japan Environment and Children's Study (n = 75,742), we evaluated maternal socioeconomic factors associated with breastfeeding 1 year after giving birth. Socioeconomic status (education, employment status, and household income), working hours, and breastfeeding were assessed using a self-administered questionnaire. After descriptive analysis, a logistic regression analysis was conducted with adjustments for age, educational level, employment status, and household income. Mothers with higher education and full-time homemakers were more likely to breastfeed one-year-old children. Mothers working long hours (both part-time and full-time) were less likely to breastfeed their one-year-old children. To improve breastfeeding among working mothers, it may be helpful to promote awareness of breastfeeding for every mother as well as to make the workplace environment conducive for working mothers to breastfeed.

Identification of LSD analogs, 1cP-AL-LAD, 1cP-MIPLA, 1V-LSD and LSZ in sheet products.

PURPOSE: Many analogs of lysergic acid diethylamide (LSD) have recently appeared as designer drugs around the world. These compounds are mainly distributed as sheet products. In this study, we identified three more newly distributed LSD analogs from paper sheet products. METHODS: The structures of the compounds were determined by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS), liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy. RESULTS: From the NMR analysis, the compounds in the four products were identified as 4-(cyclopropanecarbonyl)-N,N-diethyl-7-(prop-2-en-1-yl)-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1cP-AL-LAD), 4-(cyclopropanecarbonyl)-N-methyl-N-isopropyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo-[4,3-fg]quinoline-9-carboxamide (1cP-MIPLA), N,N-diethyl-7-methyl-4-pentanoyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1V-LSD) and (2'S,4'S)-lysergic acid 2,4-dimethylazetidide (LSZ). In comparison with the structure of LSD, 1cP-AL-LAD was converted at the positions at N1 and N6, and 1cP-MIPLA was converted at the positions at N1 and N18. The metabolic pathways and biological activities of 1cP-AL-LAD and 1cP-MIPLA have not been reported. CONCLUSIONS: This is the first report showing that LSD analogs that were converted at multiple positions have been detected in sheet products in Japan. There are concerns about the future distribution of sheet drug products containing new LSD analogs. Therefore, the continuous monitoring for newly detected compounds in sheet products is important.