Peroxisomal 4-coumaroyl-CoA ligases participate in shikonin production in Lithospermum erythrorhizon.

4-Coumaroyl-CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, which is involved in the biosynthesis of various specialized metabolites such as flavonoids, coumarins, lignans, and lignin. Plants have several 4CLs showing divergence in sequence: class I 4CLs involved in lignin metabolism, class II 4CLs associated with flavonoid metabolism, and atypical 4CLs and 4CL-like proteins of unknown function. Shikonin, a Boraginaceae-specific specialized metabolite in red gromwell (Lithospermum erythrorhizon), is biosynthesized from p-hydroxybenzoic acid, and the involvement of 4CL in its biosynthesis has long been debated. In this study, we demonstrated the requirement of 4CL for shikonin biosynthesis using a 4CL-specific inhibitor. In silico analysis of the L. erythrorhizon genome revealed the presence of at least eight 4CL genes, among which the expression of three (Le4CL3, Le4CL4, and Le4CL5) showed a positive association with shikonin production. Phylogenetic analysis indicated that Le4CL5 belongs to class I 4CLs, while Le4CL3 and Le4CL4 belong to clades that are distant from class I and class II. Interestingly, both Le4CL3 and Le4CL4 have peroxisome targeting signal 1 in their C-terminal region, and subcellular localization analysis revealed that both localize to the peroxisome. We targeted each of the three Le4CL genes by CRISPR/Cas9-mediated mutagenesis and observed remarkably lower shikonin production in Le4CL3-ge and Le4CL4-ge genome-edited lines compared with the vector control. We therefore conclude that peroxisomal Le4CL3 and Le4CL4 are responsible for shikonin production and propose a model for metabolite-specific 4CL distribution in L. erythrorhizon.

A simple method to shorten the apparent dead time in the dosimetry of Lu-177 for targeted radionuclide therapy using a gamma camera.

BACKGROUND: The dead-time loss reportedly degrades the accuracy of dosimetry using a gamma camera for targeted radionuclide therapy with Lu-177; therefore, the dead-time loss needs to be corrected. However, the correction is challenging. In this study, we propose a novel and simple method to shorten the apparent dead time rather than correcting it through experiments and Monte Carlo simulations. METHODS: An energy window of 208 keV ± 10 % is generally used for the imaging of Lu-177. Lower-energy gamma photons and X-rays of Lu-177 do not contribute to image formation but lead to dead-time losses. In our proposed method, a thin lead sheet was used to shield gamma photons and X-rays with energies lower than 208 keV, while detecting 208 keV gamma photons that penetrated the thin sheet. We measured and simulated the energy spectra and count rate characteristics of a clinical gamma camera system using a cylindrical phantom filled with a Lu-177 solution. Lead sheets of 1.0- and 0.5-mm thicknesses were used as thin shields, and the dead-time losses in tumour imaging with consumed Lu-177 were simulated. RESULTS: The apparent dead times with lead sheets of 1.0- and 0.5-mm thicknesses and without a lead sheet were 1.7, 1.9, and 5.8 µs for an energy window of 208 keV ± 10 %, respectively. The dead-time losses could be reduced from 10 % to 1.3 % using the 1.0-mm thick lead sheet in the simulated imaging of tumour. CONCLUSION: Our method is promising in clinical situations and studies on Lu-177 dosimetry for tumours.

A triple-imaging-modality system for simultaneous measurements of prompt gamma photons, prompt x-rays, and induced positrons during proton beam irradiation.

Objective. Prompt gamma photon, prompt x-ray, and induced positron imaging are possible methods for observing a proton beam's shape from outside the subject. However, since these three types of images have not been measured simultaneously nor compared using the same subject, their advantages and disadvantages remain unknown for imaging beam shapes in therapy. To clarify these points, we developed a triple-imaging-modality system to simultaneously measure prompt gamma photons, prompt x-rays, and induced positrons during proton beam irradiation to a phantom.Approach. The developed triple-imaging-modality system consists of a gamma camera, an x-ray camera, and a dual-head positron emission tomography (PET) system. During 80 MeV proton beam irradiation to a polymethyl methacrylate (PMMA) phantom, imaging of prompt gamma photons was conducted by the developed gamma camera from one side of the phantom. Imaging of prompt x-rays was conducted by the developed x-ray camera from the other side. Induced positrons were measured by the developed dual-head PET system set on the upper and lower sides of the phantom.Main results. With the proposed triple-imaging-modality system, we could simultaneously image the prompt gamma photons and prompt x-rays during proton beam irradiation. Induced positron distributions could be measured after the irradiation by the PET system and the gamma camera. Among these imaging modalities, image quality was the best for the induced positrons measured by PET. The estimated ranges were actually similar to those imaged with prompt gamma photons, prompt x-rays and induced positrons measured by PET.Significance. The developed triple-imaging-modality system made possible to simultaneously measure the three different beam images. The system will contribute to increasing the data available for imaging in therapy and will contribute to better estimating the shapes or ranges of proton beam.

Development of an event-by-event based Li-ZnS(Ag) neutron imaging detector with selective neutron detection capability.

High sensitivity and high resolution is desired in such technologies as neutron radiography. However, the contamination of gamma photons in neutron images decreases the accuracy of neutron radiography. To solve this problem, we developed an event-by-event based neutron imaging system that can selectively detect neutrons. The developed neutron imaging system consists of an Li-ZnS(Ag) scintillator plate optically coupled to a flat panel photomultiplier tube (FP-PMT) with a light guide. Scintillation light emitted from the Li-ZnS(Ag) by the interaction with neutron-induced particles is used to calculate the position based on the center of mass calculations. The spatial resolution of the neutron imaging detector is ∼2.3 mm FWHM, and the sensitivity for 252Cf at 2 cm from the source with 2-cm-thick polystyrene is 20 cps/MBq. Background count fraction from 60Co gamma photons was 0.2 %. For various types of neutron absorption phantoms, high-contrast and high-resolution neutron images are obtained with the developed neutron imaging detector using a252Cf neutron source.

A comparative study of EM-CCD and CMOS cameras for particle ion trajectory imaging.

High-resolution and real-time imaging of particle ion trajectories is essential in nuclear medicine and nuclear engineering. One potential method to achieve high-resolution real-time trajectory imaging of particle ions involves utilizing an imaging system that integrates a scintillator plate with a magnifying unit and a cooled electron multiplying charge-coupled device (EM-CCD) camera. However, acquiring an EM-CCD camera might prove challenging due to the discontinuation of CCD sensor manufacturing by vendors. As an alternative imaging approach, a low-noise, high-sensitivity camera utilizing a cooled complementary metal-oxide-semiconductor (CMOS) sensor offers a promising solution for imaging particle ion trajectories. Yet, it remains uncertain whether CMOS-based cameras can perform as effectively as CCD-based cameras in capturing particle ion trajectories. To address these concerns, we conducted a comparative analysis of the imaging performance between a CMOS-based system and an EM-CCD-based system for capturing alpha particle trajectories. The results revealed that both systems could image the trajectories of alpha particle, but the spatial resolution with the CMOS-based camera exceeded that of the EM-CCD-based camera, primarily due to the smaller pixel size of the sensor. While the signal-to-noise ratio (SNR) of the trajectory image from the CMOS-based camera initially lagged behind that from the EM-CCD-based camera, this disparity was mitigated by implementing binning techniques on the CMOS-based camera images. In conclusion, our findings suggest that a cooled CMOS camera could serve as a viable alternative for imaging particle ion trajectories.

Estimating blurless and noise-free Ir-192 source images from gamma camera images for high-dose-rate brachytherapy using a deep-learning approach.

Objective. Precise monitoring of the position and dwell time of iridium-192 (Ir-192) during high-dose-rate (HDR) brachytherapy is crucial to avoid serious damage to normal tissues. Source imaging using a compact gamma camera is a potential approach for monitoring. However, images from the gamma camera are affected by blurring and statistical noise, which impact the accuracy of source position monitoring. This study aimed to develop a deep-learning approach for estimating ideal source images that reduce the effect of blurring and statistical noise from experimental images captured using a compact gamma camera.Approach. A double pix2pix model was trained using the simulated gamma camera images of an Ir-192 source. The first model was responsible for denoising the Ir-192 images, whereas the second model performed super resolution. Trained models were then applied to the experimental images to estimate the ideal images.Main results. At a distance of 100 mm between the compact gamma camera and the Ir-192 source, the difference in full width at half maximum (FWHM) between the estimated and actual source sizes was approximately 0.5 mm for a measurement time of 1.5 s. This difference has been improved from approximately 2.7 mm without the use of DL. Even with a measurement time of 0.1 s, the ideal images could be estimated as accurately as in the 1.5 s measurements. This method consistently achieved accurate estimations of the source images at any position within the field of view; however, the difference increased with the distance between the Ir-192 source and the compact gamma camera.Significance. The proposed method successfully provided estimated images from the experimental images within errors smaller than 0.5 mm at 100 mm. This method is promising for reducing blurring and statistical noise from the experimental images, enabling precise real-time monitoring of Ir-192 sources during HDR brachytherapy.

Hybrid imaging of prompt x-rays and induced positrons using a pinhole gamma camera during and after irradiation of protons.

Objective. Prompt x-ray imaging using a low-energy x-ray camera is a promising method for observing a proton beam's shape from outside the subject. Furthermore, imaging of positrons produced by nuclear reactions with protons is a possible method for observing the beam shape. However, it has not been possible to measure these two types of images with a single imaging system due to the limited imaging capability of existing systems. Imaging of both prompt x-rays and the distribution of positrons may compensate for the shortcomings of each method.Approach. We conducted imaging of the prompt x-ray using a pinhole x-ray camera during irradiation with protons in list mode. Then, after irradiation with protons, imaging of annihilation radiations from the produced positrons was conducted using the same pinhole x-ray camera in list mode. After this imaging, list-mode data were sorted to obtain prompt x-ray images and positron images.Main results. With the proposed procedure, we could measure both prompt x-ray images and induced positron images with a single irradiation by a proton beam. From the prompt x-ray images, ranges and widths of the proton beams could be estimated. The distributions of positrons were slightly wider than those of the prompt x-rays. From the time sequential positron images, we could derive the time activity curves of the produced positrons.Significance. Hybrid imaging of prompt x-rays and induced positrons using a pinhole x-ray camera was achieved. The proposed procedure would be useful for measuring prompt x-ray images during irradiation to estimate the beam structures as well as for measuring the induced positron images after irradiation to estimate the distributions and time activity curves of the induced positrons.

Comparison of organ dose from chest radiography with varying beam quality and constant exposure index.

The optimum X-ray tube voltage for chest radiographic examinations remains unclear; hence, the tube voltage varies between medical facilities. An exposure index (EI) was proposed to standardize the parameters for radiographic examinations. However, even if identical EI values are used to examine the same person, organ doses may vary due to differences in tube voltages. In this study, the variation in organ doses between different beam qualities under identical EI values for chest radiographic examinations was investigated using Monte Carlo simulations. A focused anti-scatter grid as well as standard and larger physique-type medical internal radiation dose (MIRD) phantoms were studied under tube voltages of 90, 100, 110, and 120 kVp. The organ doses in the MIRD phantom increased as the X-ray tube voltage decreased, even with identical EI values. The absorbed doses in the lungs of standard and large-sized MIRD phantoms at 90 kVp were 23% and 35% higher than those at 120 kVp, respectively. The doses to organs other than the lung at 90 kVp were also higher than those at 120 kVp. From the perspective of reducing radiation doses, a tube voltage of 120 kVp is considered better for chest examinations compared with a tube voltage of 90 kVp under identical EI values.

Development of an ultrahigh resolution real time alpha particle imaging system for observing the trajectories of alpha particles in a scintillator.

High-resolution imaging of alpha particles is required in the detection of alpha radionuclides in cells or small organs for the development of radio-compounds for targeted alpha-particle therapy or other purposes. We developed an ultrahigh resolution, real time alpha-particle imaging system for observing the trajectories of alpha particles in a scintillator. The developed system is based on a magnifying unit and a cooled electron multiplying charge-coupled device (EM-CCD) camera, combined with a 100-µm-thick Ce-doped Gd3Al2Ga3O12 (GAGG) scintillator plate. Alpha particles from an Am-241 source were irradiated to the GAGG scintillator and imaged with the system. Using our system, we measured the trajectories of the alpha particles having different shapes in real time. In some of these measured trajectories, the line shapes of the alpha particles that flew in the GAGG scintillator were clearly observed. The lateral profiles of the alpha-particle trajectories were imaged with widths of ~ 2 µm. We conclude that the developed imaging system is promising for research on targeted alpha-particle therapy or other alpha particle detections that require high spatial resolution.

Optimization of the energy window setting in Ir-192 source imaging for high-dose-rate brachytherapy using a YAP(Ce) gamma camera.

PURPOSE: Although real-time imaging of the high-activity iridium-192 (Ir-192) source position during high-dose-rate (HDR) brachytherapy using a high-energy gamma camera system is a promising approach, the energy window was not optimized for spatial resolution or scatter fraction. METHODS: By using a list-mode data-acquisition system that can acquire energy information of a cerium-doped yttrium aluminum perovskite (YA1O3: YAP(Ce)) gamma camera, we tried to optimize the energy window's setting to improve the spatial resolution and reduce scatter fraction. RESULTS: The spatial resolution was highest for the central energy of the window at ∼300 keV. The scatter fraction was also smallest for the central energy of the window at ∼300 keV, and the scatter fraction was more than 48 % smaller than that for the full energy window. CONCLUSIONS: We clarified that the spatial resolution can be improved and the scatter fraction can be reduced through optimizing the energy window of the YAP(Ce) gamma camera by setting the central energy of the window to ∼300 keV for HDR brachytherapy.

Technical note: Short-time sequential high-energy gamma photon imaging using list-mode data acquisition system for high-dose-rate brachytherapy.

PURPOSE: Measurement of the dwell time and moving speed of a high-activity iridium-192 (Ir-192) source used for high-dose-rate (HDR) brachytherapy is important for estimating the precise dose delivery to a tumor. For this purpose, we used a cerium-doped yttrium aluminum perovskite (YA1O3 :YAP(Ce)) gamma camera system, combined with a list-mode data acquisition system that can acquire short-time sequential images, and measured the dwell times and moving speeds of the Ir-192 source. METHODS: Gamma photon imaging was conducted using the gamma camera in list mode for the Ir-192 source of HDR brachytherapy with fixed dwell times and positions. The acquired list-mode images were sorted to millisecond-order interval time sequential images to evaluate the dwell time at each position. Time count rate curves were derived to calculate the dwell time at each source position and moving speed of the source. RESULTS: We could measure the millisecond-order time sequential images for the Ir-192 source. The measured times for the preset dwell times of 2 s and 10 s were 1.98 to 2.00 s full width at half maximum (FWHM) and 10.0 s FWHM, respectively. The dwell times at the first dwell position were larger than those at other positions. We also measured the moving speeds of the source after the dwells while moving back to the afterloader and found the speed increased with the distance from the edge of the field of view to the last dwell position. CONCLUSION: We conclude that millisecond-order time sequential imaging of the Ir-192 source is possible by using a gamma camera and is useful for evaluating the dwell times and moving speeds of the Ir-192 source.

Mechanism of Budded Virus Envelope Fusion into a Planar Bilayer Lipid Membrane on a SiO2 Substrate.

Artificial planar bilayer lipid membranes (BLMs) are simple models of cellular systems under physically and chemically controlled conditions, and they have been used to investigate membrane protein activity. Baculovirus-budded virus (BV) systems can express recombinant membrane proteins. In this study, aiming for membrane protein reconstitution, we examined the fusion of BVs containing recombinant membrane proteins into artificial planar BLMs on a Si microwell substrate. BV fusion with the BLMs depended on the pH of the solution, and it was enhanced at lower pH. Based on fluorescence recovery after photobleaching (FRAP) measurement, the fusion state of BVs was evaluated, and full fusion at low pH was confirmed. The fluorescent labeling the membrane proteins was also observed in the freestanding part of the BLMs as well as in the supported part. These results demonstrate the effectiveness of BLMs as a platform to examine detailed fusion dynamics of BVs. Furthermore, this study revealed that the fusion of BVs is a promising method for reconstituting membrane proteins to artificial freestanding BLMs for the development of biodevices with which we can examine membrane protein activity.

Performance evaluation of YAlO3 scintillator plates with different Ce concentrations.

Since Ce-doped YAlO3 (YAP(Ce)) scintillators have small non-proportionality, it is useful to develop a radiation imaging detector for low-energy gamma photons or X-rays. However, the YAP(Ce) performance with different Ce concentrations remains unclear. Consequently, we measured the basic performance of YAP(Ce) plates with different Ce concentrations. We used three types of YAP(Ce) scintillator plates with different Ce concentrations: 0.05% Ce, 1% Ce, and 2% Ce. The YAP(Ce) plates were 10 mm × 10 mm x 0.5 mm. We measured and compared the energy spectra, the decay times, the α-γ ratio, and the non-proportionality. We also evaluated the relation between these performances and the Ce concentrations. The light output of a YAP(Ce) showed positive correlation with the Ce concentrations, and the decay time of the YAP(Ce) showed a negative correlation with them. The energy resolution slightly improved for a YAP(Ce) with higher Ce concentrations. We found the α-γ ratio were slightly larger for higher Ce concentrations. The YAP(Ce) plate with a 2% Ce had the highest light output and the best energy resolution as well as the shortest decay time. Based on these results, the YAP(Ce) plate with a concentration of 2% Ce is a better selection for the development of radiation detectors or radiation imaging detectors for low-energy gamma photons or X-rays as well as alpha particles.

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon.

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.

Membrane fusion and infection abilities of baculovirus virions are preserved during freezing and thawing in the presence of trehalose.

Budded viruses (BVs) of baculovirus such as Autographa californica nucleopolyhedrovirus (AcNPV) have recently been studied as biological nanomaterials, and methods for their longer-term storage without deterioration would be desirable. The cryopreservation of virions with a naturally occurring saccharide like trehalose as a cryoprotectant is known to be useful for maintaining the viral structure and function. In this study, we examined how useful trehalose is as protectant for BV cryopreservation during repeated freeze-thaw cycles: 1) membrane fusion between liposomes (multilamellar vesicles, MLVs) and BVs, 2) infection of insect culture cells (Sf9 cells) by RFP-expressing BVs, and 3) morphologies of these BVs were investigated by fluorescent dequenching assay, fluorescence microscopy, and transmission electron microscopy (TEM), respectively. The results suggest that the BVs deteriorate in quality with each freeze-thaw cycle, and this deterioration can be diminished with the use of trehalose to an extent similar to that seen with storage on ice.

Adhesion of Epoxy Resin with Hexagonal Boron Nitride and Graphite.

Adhesion interaction of epoxy resin with the basal surfaces of h-BN and graphite is investigated with the first-principles density functional theory calculations in conjunction with the dispersion correction. The h-BN/epoxy and graphite/epoxy interfaces play an important role in producing nanocomposite materials with excellent thermal dissipation properties. The epoxy resin structure is simulated by using four kinds of fragmentary models. Their structures are optimized on the h-BN and graphite surfaces after an annealing simulation. The distance between the epoxy fragment and the surface is about 3 Å. At the interface between h-BN and epoxy resin, no H-bonding formation is observed, though one could expect that the active functional groups of epoxy resin, such as hydroxyl (-OH) group, would be involved in a hydrogen-bonding interaction with nitrogen atoms of the h-BN surface. The adhesion energies for the two interfaces are calculated, showing that these two interfaces are characterized by almost the same strength of adhesion interaction. To obtain the adhesion force-separation curve for the two interfaces, the potential energy surface associated with the detachment of the epoxy fragment from the surface is calculated with the help of the nudged elastic band method and then the adhesion force is obtained by using either the Morse-potential approximation or the Hellmann-Feynman force calculation. The results from both methods agree with each other. The maximum adhesion force for the h-BN/epoxy interface is as high as that for the graphite/epoxy interface. To better understand this result, a force-decomposition analysis is carried out, and it has been disclosed that the adhesion forces working at both interfaces mainly come from the dispersion force. The trend of increase in the C 6 parameters used for the dispersion correction for the atoms included in the h-BN or graphite surface is in the order: N < C < B, which reasonably explains why the strengths of the dispersion forces operating at the two interfaces are similar. Also, the electron localization function analysis can explain why the h-BN surface cannot form an H bond with the hydroxyl group in epoxy resin.

The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins.

Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.

Effects of short term fasting on the expression of genes involved in lipid metabolism in chicks.

The aim of this study was to analyze the expression patterns of key genes involved in lipid metabolism in response to short term fasting in chicks (Gallus gallus). The mRNA level of the genes was analyzed after 0, 2, and 4 h of fasting in the liver and white adipose tissue. In the liver, the mRNA level of peroxisome proliferator-activated receptor α was significantly increased after 2 h of fasting. The mRNA levels of carnitine palmitoyltransferase 1a and acyl-CoA oxidase were significantly increased after 4 h of fasting. In contrast, the mRNA levels of sterol regulatory element-binding protein 1, acetyl-CoA carboxylase α, and fatty acid synthase were significantly decreased after 4 h of fasting. The mRNA levels of cholesterol metabolism-related genes such as 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7α-hydroxylase were significantly decreased after 4 h of fasting. In the white adipose tissue, the mRNA level of adipose triglyceride lipase was significantly increased after 4 h of fasting. In contrast, the mRNA levels of peroxisome proliferator-activated receptor γ and lipoprotein lipase were significantly decreased after 4 h of fasting. These results demonstrated that the gene expression of lipid metabolism-related genes is regulated by short term fasting in both the liver and WAT in chicks.

Synthetic studies on kohamaic acids: synthesis of structurally simplified analogs of kohamaic acid A.

Kohamaic acid A is a potent DNA polymerase inhibitor isolated from the Okinawan marine sponge Ircinia sp. A series of structurally simplified analogs of kohamaic acid A were synthesized with the aim of evaluating structure-activity relationships.