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Abstract The impact of 2-ethylanthraquinone (2-EAQ) on alkaline cooking of Eucalyptus pellita wood was examined by analyzing pulp yield, kappa number, the structure and weight-averaged molecular weight (Mw) of dissolved lignin in black liquor and residual lignin in pulp (RL). During soda cooking under 23% of active alkaline (AA) condition, adding 0.06% 2-EAQ reduced the kappa number by 12 points compared to cooking without it. The addition of 0.06% 2-EAQ to kraft cooking increased pulp yield by about 4% at about 19 of kappa number compared to the no addition. The analysis of lignin structure by using semiquantitative heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR) showed that the addition of 2-EAQ promoted β-ether cleavage during alkaline cooking and reduced peeling reaction of polysaccharides and prevention of the elimination of γ-OH of lignin to form enol ethers and stilbenes. Overall, the addition of 2-EAQ promoted the degradation of lignin to lower molecular weight compounds, with a more pronounced effect observed in soda cooking than in kraft cooking. Under optimal conditions, the delignification efficiency and pulp yield of soda-EAQ pulping were comparable to kraft cooking.

Abstract The old Ginkgo biloba L. trees often develop cylindrical woody structures that grow downward from the underside of the large branches near the trunks. This structure is traditionally called “chi-chi” (a breast) in Japan. The structure of chi-chi has not been investigated in detail because of the rarity of such old trees of G. biloba. This study examined the chi-chi from wood anatomy and chemistry viewpoints. After debarking, there were many woody bulges and latent buds. There were hollows corresponding to these latent buds on the inner side of the bark. In the transverse section obtained from the tip part of the chi-chi sample, we found tracheids curving in a horizontal plane, and the parenchymatous latent bud tissue is the center of the swirl. Microscopic observations and X-ray micro-computed tomography suggest the growing course of the chi-chi due to several swirls contiguous to each other. From these observations, the downward growth of the chi-chi starting from the cambium cell division might be driven by the woody bulges with latent buds. The cell walls of the curved tracheids were not thickened, not rounded, and had no intercellular spaces, but their S3 layers disappeared. Furthermore, the results of thioacidolysis and acetyl bromide analyses exhibited p-hydroxyphenyl subunits deposition to lignin and high lignin content on the tip part of the chi-chi. These results suggest that the curved tracheids in the chi-chi have compression-wood-like properties to some extent.

Abstract The lignocellulosic biorefinery industry can be an important contributor to achieving global carbon net zero goals. However, low valorization of the waste lignin severely limits the sustainability of biorefineries. Using a hydrothermal reaction, we have converted sulfuric acid lignin (SAL) into a water-soluble hydrothermal SAL (HSAL). Here, we show the improvement of HSAL on plant nutrient bioavailability and growth through its metal chelating capacity. We characterize HSAL’s high ratio of phenolic hydroxyl groups to methoxy groups and its capacity to chelate metal ions. Application of HSAL significantly promotes root length and plant growth of both monocot and dicot plant species due to improving nutrient bioavailability. The HSAL-mediated increase in iron bioavailability is comparable to the well-known metal chelator ethylenediaminetetraacetic acid. Therefore, HSAL promises to be a sustainable nutrient chelator to provide an attractive avenue for sustainable utilization of the waste lignin from the biorefinery industry.

IntroductionPhellodendron amurense Rupr. contains rich alkaloids, which have been extensively applied in clinical treatments for their various biological activities. However, detailed microscopic distribution and roles of such alkaloids in P. amurense stem still need to be clarified.MethodsIn this study, the distribution of eight alkaloids in the transverse surface of freeze-fixed P. amurense stems in fall and summer has been visualized by cryo-time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM), which was found in living tissues with relative contents of different alkaloids varying with the position. In addition, the contents of these alkaloids quantified by high-performance liquid chromatography (HPLC) analysis suggested the seasonal variation from fall to the following summer.Results and discussionDistribution of eight alkaloids in the freeze-fixed stems of P. amurense from fall and summer seasons has been visualized and assigned into specific living tissues, with relative contents varying in different positions with seasons, which suggested their possible roles in the physiological processes of the plant itself or plant responding to changes in the surrounding conditions.ConclusionThis study provided a significant basis for further discussion of the genes or enzymes involved in these processes, which will contribute to investigating biosynthetic pathways and specific in planta roles of alkaloids.

Abstract Some species of Diospyros form the black wood that is known as ebony. Infrequently, D. kaki forms wood with black patterning that is called “kurogaki” in Japan and is highly valued. To understand the mechanism of formation of the blackened xylem, we attempted to clarify the pathways of diffusion of colored substances from the site of their biosynthesis to their deposition in secondary xylem. We observed colored substances in pits and intercellular spaces and we recorded the deposition of colored substances in the cell walls of wood fibers. In gray and black regions, we found colored substances in the pits between xylem cells, in blind pits and in intercellular spaces. At the boundary between whitish and black regions, areas of coloration in the cell walls of wood fibers gradually increased in size from the whitish to the black regions. Heavy deposition of colored substances was observed in the inner region of the secondary walls of wood fibers. Furthermore, coloration of the outer region of the secondary walls and compound middle lamella (CML) of wood fibers gradually became stronger toward the black regions. Our observations suggest that pit-pairs and intercellular spaces might provide the pathways for inter- and extracellular diffusion of colored substances, respectively. In addition, colored substances might penetrate the cell walls of wood fibers, moving from the lumen into the cell wall.

Abstract In many flowering plants, floral scents are a significant trait for visitors, playing an important role in attracting pollinators and/or detracting herbivores. The evolution of flowering plants from hermaphroditism to dioecy is often accompanied by sexual dimorphism in floral scent. In this study, floral scents emitted by different sexual morphs of the subdioecious shrub Eurya japonica Thunb. were collected using a dynamic headspace method, and sexual and temporal variations were evaluated by gas chromatography–mass spectrometry (GC–MS). Two volatiles, α‐pinene and linalool, were identified as the major components of floral scents in females, hermaphrodites, and males. The males emit higher amounts of floral scents, particularly α‐pinene, compared to females or hermaphrodites. Floral scents emitted by males generally decrease as flowers enter senescence, whereas those from females or hermaphrodites do not significantly differ. Intraspecific variations in floral scents of subdioecious species provided by this study would contribute to better understanding of sexual dimorphism in floral scent.

Abstract The purpose of the present study is to clarify the diffusion of non-volatile substances into cell walls during the conditioning procedure under varying relative humidities (RH). In this paper, wood blocks were impregnated using an aqueous solution of melamine formaldehyde (MF), and they were subsequently conditioned under RHs of 11, 43, and 75%. The solute that diffused into the cell walls was visualized using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The volumetric relative swelling of the samples during the conditioning procedure was calculated. The results showed increased cell wall swelling at higher RH, which may have been caused by higher MF diffusion into the cell walls and/or higher moisture content. Cryo-TOF-SIMS measurements showed that more cell cavities were unfilled with MF at higher RH, indicating that most of the MF diffused from the cell cavities into the cell walls. The relative intensity of MF in the cell walls of the cured samples was evaluated from dry-TOF-SIMS images, which showed a higher relative intensity of MF in the cell walls at higher RH. With the ability to visualize and semi-quantitatively evaluate the solute in cell walls, TOF-SIMS will serve as a powerful tool for future studies of solute diffusion mechanisms in solution-impregnated wood.

Lignin is the second most abundant natural polymer on Earth and is a major cell wall component in vascular plants. Lignin biosynthesis has three stages: biosynthesis, transport, and polymerization of its precursors. However, there is limited knowledge on lignin precursor transport, especially in monocots. In the present study, we aimed to elucidate the transport mode of lignin monomers in the lignifying tissues of bamboo (Phyllostachys pubescens). The growth manners and lignification processes of bamboo shoots were elucidated, which enabled us to obtain the lignifying tissues reproducibly. Microsomal membrane fractions were prepared from tissues undergoing vigorous lignification to analyze the transport activities of lignin precursors in order to show the ATP-dependent transport of coniferin and p-glucocoumaryl alcohol. The transport activities for both precursors depend on vacuolar type H+-ATPase and a H+ gradient across the membrane, suggesting that the electrochemical potential is the driving force of the transport of both substrates. These findings are similar to the transport properties of these lignin precursors in the differentiating xylem of poplar and Japanese cypress. Our findings suggest that transport of coniferin and p-glucocoumaryl alcohol is mediated by secondary active transporters energized partly by the vacuolar type H+-ATPase, which is common in lignifying tissues. The loading of these lignin precursors into endomembrane compartments may contribute to lignification in vascular plants.

Abstract Alkaloids are basic nitrogen-containing chemicals that have important physiological and pharmacological characteristics. Many vascular plant species contain alkaloids, and their roles in planta are of interest. However, the detailed distribution of alkaloids remains unclear because of their low water solubility and low concentrations in plants. In this study, we visualized the distribution of salicifoline, a water-soluble quaternary ammonium alkaloid, in the freeze-fixed stems of Magnolia kobus by cryo time-of-flight secondary ion mass spectrometry. Most of the salicifoline was distributed in living phloem tissues. In the xylem, salicifoline was detected in ray cells, lignifying wood fibres, and in vessels in the latest annual ring. The salicifoline distribution in the xylem varied with the cell wall formation stage. These results provide new insights into the storage, transportation, and role of the alkaloid salicifoline in M. kobus.

Abstract Background Orthopedic surgeries of lower extremities such as total knee arthroplasty (TKA), total hip arthroplasty (THA), and hip fracture surgery (HFS) are widely considered to carry a high risk of developing deep venous thrombosis (DVT) and pulmonary thromboembolism (PTE). Growing attention to epidemiological studies using a healthcare database led us to quantify the risks using a Japanese database to reveal recent medical care for such events. Methods The study comprised 36,947 patients who had undergone orthopedic surgeries of the lower extremities and whose medical information from April 2008 to September 2013 was available. The source population of the database was derived from 100 acute-care hospitals with Diagnosis Procedure Combination. The events were defined by diagnosis, medication, imaging, and laboratory tests. Results A breakdown of patients who underwent orthopedic surgeries by type of surgery showed 13.6% for TKA, 10.4% for THA, 56.8% for HFS, 1.5% for rupture of Achilles tendon, and 18.0% for simple fracture of lower extremities. The incidence for DVT, PTE, and bleeding events by type of surgery was 1.3, 0.2, and 1.0% for TKA; 0.9, 0.2, and 1.1% for THA; and 0.4, 0.1, and 1.8% for HFS, respectively. The population for risk factor analysis consisted of patients with similar background factors who underwent TKA, THA, or HFS. The statistically significant risk factors for PTE analyzed by the backward elimination procedure in a multivariate model were female sex, history of venous thromboembolism (VTE), thrombophilia, and varicose veins of lower extremity. Conclusions The incidence of DVT, PTE, and bleeding events and the risk factors for DVT and PTE in patients by type of orthopedic surgeries of lower extremities found in our study are considered to be rational as they reflect evidence from real-world cases. Trial registration University Hospital Medical Information Network Clinical Trials Registry UMIN000012667

Abstract Monolignols are precursors of lignin, and their glucosides are often found in plants. Glucosylation creates water‐soluble and chemically stable monolignols by protecting the phenolic hydroxyl group. To discuss the role of sinapyl alcohol glucoside, syringin, in planta, the cellular distribution of syringin in the transverse and radial surfaces of quick‐frozen stems of Syringa vulgaris L. (lilac) was visualized by cryo‐time‐of‐flight secondary ion mass spectrometry and scanning electron microscopy (cryo‐TOF‐SIMS/SEM) analyses. The amount and rough distribution of syringin were confirmed by high‐performance liquid chromatography measurements using serial tangential sections of freeze‐fixed lilac stems. The syringin distribution was also discussed with reference to the tissue classification from microscopic observations. Syringin was mainly found in the phloem region. In the xylem region, syringin was evenly distributed irrespective of the cell type from the cambial zone to the early differentiating stage region and selectively distributed in vessels in the later differentiating stage region. After the lignification of wood fibers, syringin was found in rays and some vessels in the initial part of the annual rings. Previously, artificially administered isotope‐labeled syringin was shown to be assimilated into lignin in the differentiating xylem region. Based on this, our present data showing syringin storage in the differentiating xylem region and its variation depending on the lignification stage suggest that syringin works as a lignin precursor. Additionally, detection of syringin in vessels and rays indicates intercellular transportation of syringin in lilac stems.

Lignin is an essential component of higher plants, which is built by the enzymatic dehydrogenative polymerization of monolignols. First, monolignol is enzymatically oxidized to produce the phenoxy radical, which can form resonance hybrids. Two radical resonant hybrids are coupled with each other to yield dilignol with various linkage types, of which the main structures are β-O-4′ (I), β-5′ (II) and β-β′ (III). However, the reaction mechanism behind the addition lignol radicals to dilignol is not yet fully understood. Here, we show an unexpected reaction with structure II during enzymatic dehydrogenative polymerization, which involves cleavage of a covalent linkage and creation of a new radical coupling site. This implied that the β-5 dilignol diversifies the growing pattern of lignin. This discovery elucidates a novel mechanism in lignin polymerization.

Electropolymerization was carried out in order to obtain a bio-based polymer by a simple procedure. Ferulic acid, a component of gramineous plants, was selected for this purpose. A thin polymer film was produced in an organic solvent medium (i.e., CH2Cl2/methanol (4:1 v/v) in the presence of 0.2 M LiClO4), whereas it was not obtained in a totally aqueous medium (0.2 M NaOH). Scanning electron microscopic analysis showed that the polymer film had a porous lamellar structure of ~10 μm thickness. Most of the carboxyl groups remained, and IR spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses revealed that o-quinones existed in the polymer film. After electropolymerization, caffeic acid, sinapic acid, 3,6-bis(4-hydroxy-3-methoxyphenyl)tetrahydrofuro[3,4-c]furan-1,4-dione (1), and 4,4’-dihydroxy-3,3’-dimethoxy-β,β’-bicinnamic acid (2) were detected in the organic reaction medium; this suggested that demethylation, methoxyl group addition, and radical coupling reactions occurred at the electrode surface.

Three-dimensional maxillary bone models of a male and a female patient were constructed using their CT-images. The distributions of Young's modulus were estimated from their bone mineral density distributions. Total six implants were embedded into each of the maxillary models. Finite element analysis of the maxilla models was then performed in order to assess the concentrations of strain energy density especially in the vicinities of the embedded implants. It was found that in both models, strain energy density was concentrated especially around the right-molar implant, suggesting outbreak of damage and subsequent absorption of bone tissue in this region. The female model with smaller size and lower bone density exhibited much higher localized concentration of strain energy density than the male model. Therefore, a modified placement of the right-molar implant was then introduced into the female model and such high concentration was effectively reduced by using the inclined and longer implant. It is thus concluded that this kind of three-dimensional modeling can clinically be used to predict the optimal implant treatment for each of dental patients.

Previously, a few models have been proposed to predict bone resorption process due to stress shielding in long bones such as proximal femur; however, there are almost no reports on finite element analysis of loss of marginal dental bone that is caused mainly by occlusive overload. In this work, the stress, strain and strain energy density (SED) criteria were separately applied to simulate overload-induced bone resorption in a jawbone/implant system by means of the finite element analysis. A simplified dental bone/implant model was created, with the bone composed of a cortical bone and a cancellous bone and the implant having the detailed screw structure. The results demonstrated that the simulations according to the equal SED criterion reproduce bone resorption patterns that are more realistic to actual clinical situations, when compared to the equal stress or strain criterion. It was shown that bone resorption starts initially in the cortical bone around the implant neck, then extends downwards, and lastly enters the cancellous bone after passing through the interface of the cortical and cancellous bone. A symmetric bone resorption pattern was revealed under the condition of axial loading, whereas an asymmetric resorption prototype was demonstrated under the oblique loading condition. Moreover, in the case of oblique loading, bone resorption is faster and the amount of resorbed bone is larger, which leads to more micromotion of the dental implant than in the case of axial loading.

A porcine mandible was separated to prepare thin periodontium specimens, consisting of a molar, periodontal ligament (PDL), and alveolar bone. Occlusion was simulated by applying a forced compressive displacement, using a table-top material tester. We photographed images of the displacing periodontium specimen and simultaneously obtained the load-displacement curve during the test. The displacement and deformation distributions were examined using digital image correlation analysis. Then, we correlated the distribution with the load-displacement curve, which was characterized by biphasic behavior, as noted in many previous studies. We found that the displacement and deformation distributions of actual periodontium correlated with the load-displacement curve during dental occlusion. Regarding the biphasic characteristics of the load-displacement curve, we showed experimentally that the first phase indicated deformation of the PDL and the second indicated deformation of the alveolar bone and tooth.

In the present study, peri-implant tissue alteration was studied histologically, and then differences in the gene expression pattern of certain proteins were analyzed using cDNA microarray analysis, under the influence of static lateral load. Beagle dogs were divided into two groups: control and load. Three months after the extraction of mandibular premolars, two implants were placed in each side of the mandible. Each group was further divided into subgroups for loading times of 1, 7 and 14 days. Immediately after the implant installation, a loading device was adhered between the abutments of neighboring implants. Afterward, the expansion screw of the load group was activated by 360-degree rotation, resulting in 0.7 mm horizontal expansion. After sacrificing the dogs, peri-implant tissue was collected, then microarray analysis for the detection of gene expression was performed, as well as histology. Histology indicated that the peri-implant tissue broke down under the influence of the load. Microarray analysis demonstrated the presence of load-sensitive gene expression even after 1 day. Differences in gene expression patterns in concert with static lateral load were demonstrated, even at an early stage post-operation. Further study may help in the diagnosis of pathological overload.

In this study, we clarified dental occlusion functions by visualizing the displacement and deformation patterns of the periodontium under dental occlusion. A digital image correlation method served as a visualization and deformation analysis method using fresh porcine periodontium specimens. Also using dried periodontium specimens, we conducted the occlusion test to characterize the differences in displacement and deformation patterns between fresh and dried specimens. Results indicated that the periodontal ligaments deform first, followed by alveolar bones. Marked differences in displacement and deformation patterns were observed between the fresh and dried periodontium specimens.