Prior secondary cell wall formation is required for gelatinous layer deposition and posture control in gravi-stimulated aspen.

Cell walls, especially secondary cell walls (SCWs), maintain cell shape and reinforce wood, but their structure and shape can be altered in response to gravity. In hardwood trees, tension wood is formed along the upper side of a bending stem and contains wood fiber cells that have a gelatinous layer (G-layer) inside the SCW. In a previous study, we generated nst/snd quadruple-knockout aspens (Populus tremula × Populus tremuloides), in which SCW formation was impaired in 99% of the wood fiber cells. In the present study, we produced nst/snd triple-knockout aspens, in which a large number of wood fibers had thinner SCWs than the wild type (WT) and some had no SCW. Because SCW layers are always formed prior to G-layer deposition, the nst/snd mutants raise interesting questions of whether the mutants can form G-layers without SCW and whether they can control their postures in response to changes in gravitational direction. The nst/snd mutants and the WT plants showed growth eccentricity and vessel frequency reduction when grown on an incline, but the triple mutants recovered their upright growth only slightly, and the quadruple mutants were unable to maintain their postures. The mutants clearly showed that the G-layers were formed in SCW-containing wood fibers but not in those lacking the SCW. Our results indicate that SCWs are essential for G-layer formation and posture control. Furthermore, each wood fiber cell may be able to recognize its cell wall developmental stage to initiate the formation of the G-layer as a response to gravistimulation.

Change in lignin structure, but not in lignin content, in transgenic poplar overexpressing the rice master regulator of secondary cell wall biosynthesis.

We previously succeeded in enhancing wood formation of wood in transgenic poplar plants by overexpressing secondary wall NAM/ATAF/CUC (NAC) domain protein 1 from Oryza sativa (OsSWN1), a transcription factor 'master regulator' of secondary cell wall formation in rice, under control of the fiber preferential NST3/SND1 promoter from Arabidopsis. Transgenic plants had an increased cell wall thickness and cell wall density of individual cells in the secondary xylem of stems as well as an increased wood density. OsSWN1 triggers the induction of polysaccharide and lignin biosynthetic gene expressions, however, resulting in no significant impact on the lignin content in the transgenic plants. In contrast, wet and dry chemical analyses of lignin revealed changes in S/G ratio and in the composition of lignin interunit linkages in transgenic lines. The results from gene expression analysis suggest that the structural changes in lignin were due to an unbalanced induction of lignin biosynthetic genes in transgenic lines. Our present data indicate that the overexpression of the chimeric transcription factor causes accelerated deposition of secondary cell wall components including lignin and polysaccharides through an acquired mechanism.

Expression divergence of cellulose synthase (CesA) genes after a recent whole genome duplication event in Populus.

MAIN CONCLUSION: Secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules. Gene duplication is an important mechanism for functional divergence of genes. Secondary cell wall-associated cellulose synthase genes (CesA4, CesA7 and CesA8) are duplicated in Populus plants due to a recent whole genome duplication event. Here, we demonstrate that duplicate CesA genes show tissue-dependent expression divergence in Populus plants. Real-time PCR analysis of Populus CesA genes suggested that Pt × tCesA8-B was more highly expressed than Pt × tCesA8-A in phloem and secondary xylem tissue of mature stem. Histochemical and histological analyses of transformants expressing a GFP-GUS fusion gene driven by Populus CesA promoters revealed that the duplicate CesA genes showed different expression patterns in phloem fibers, secondary xylem, root cap and leaf trichomes. We predicted putative cis-regulatory motifs that regulate expression of secondary cell wall-associated CesA genes, and identified 19 motifs that are highly conserved in the CesA gene family of eudicotyledonous plants. Furthermore, a transient transactivation assay identified candidate transcription factors that affect levels and patterns of expression of Populus CesA genes. The present study reveals that secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules.

The promoter of an A9 homolog from the conifer Cryptomeria japonica imparts male strobilus-dominant expression in transgenic trees.

KEY MESSAGE : GUS analysis in Cryptomeria japonica revealed that the CjMALE1 promoter is activated in the male strobilus of C. japonica. Toward the development of male sterile technology for Cryptomeria japonica, a male strobilus-dominant promoter of C. japonica was isolated. The CjMALE1 gene was isolated from a male strobilus-specific suppression subtractive hybridization (SSH) library, and the promoter was isolated by the TAIL-PCR method. To characterize the CjMALE1 promoter, ß-glucuronidase (GUS)-fused genes were constructed and introduced into C. japonica using Agrobacterium tumefaciens. GUS expression from CjMALE1-2.5 K (2,718 bp fragment)::GUS C. japonica and CjMALE1-1 K (1,029 bp fragment)::GUS C. japonica was detected in the tapetum and microspore mother cells. These promoter fragments were comparably active in the pre-meiotic stage of the male strobilus of C. japonica. Our analysis showed that the 1,029 bp promoter had all the cis-elements necessary for male strobilus-dominant expression of CjMALE1. When CjMALE1-1 K::GUS was introduced into Arabidopsis, GUS expression was detected in the same spatiotemporal pattern as in C. japonica. These results suggest that the CjMALE1 promoter is subject to transcriptional regulatory systems consisting of cis- and trans-elements that have been highly conserved during evolution.

Microscopic distribution of alkaloids in freeze-fixed stems of Phellodendron amurense.

Introduction: Phellodendron 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. Methods: In 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 discussion: Distribution 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. Conclusion: This 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.

Highly potent, orally active novel small-molecule HPK1 inhibitor DS21150768 induces anti-tumor responses in multiple syngeneic tumor mouse models.

Augmenting T-cell activity is a promising approach to enhance the efficacy of cancer immunotherapy treatment. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in immune cells and negatively regulates T-cell receptor signaling. It is reported that inhibition of the kinase function of HPK1 results in tumor growth suppression by enhancing cancer immunity. Thus, developing HPK1 inhibitors has attracted considerable attention as a future cancer immunotherapy approach. However, despite recent progress in HPK1 biology and pharmacology, various challenges still remain, such as developing HPK1 inhibitors with favorable pharmacological profiles and identifying tumor characteristics that can be applied to define susceptibility to HPK1 inhibition. Here, we present the identification and pharmacological evaluation of DS21150768, a potent small-molecule HPK1 inhibitor with a novel chemical scaffold. DS21150768 shows remarkable inhibition of HPK1 kinase activity, and in vitro studies demonstrated its potent activity to enhance T-cell function. DS21150768 is orally bioavailable and shows sustained plasma exposure, which leads to enhanced cytokine responses in vivo. We conducted a comparison of the anti-tumor efficacy of DS21150768 alone or in combination with anti-PD-1 antibody in 12 different mouse cancer cell models, and observed that the treatments suppressed tumor growth in multiple models. Furthermore, Gene Set Enrichment Analysis demonstrated significant enrichment of immune-related gene signatures in the tumor models responsive to DS21150768 treatment. Our results provide a path forward for the future development of HPK1 inhibitors and fundamental insights into biomarkers of HPK1-targeted therapy.

CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.

CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don).

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

Endoscopic removal of accidental aspirated and ingested dental foreign bodies: A cross-sectional study.

ABSTRACT: The incidence of accidental ingestion and aspiration of foreign body (FB) is likely to occur. Many FBs are discharged spontaneously, but many dental FBs are often sharp and may remain in the pharynx, esophagus, and stomach, causing serious complications such as hemorrhage, asphyxia, perforation of the digestive tract, mediastinal emphysema, peritonitis, and ileus. We aimed to examine which type of dental foreign bodies can be removed by endoscope.In this study, we enrolled 32 patients who were evaluated at the Emergency and Critical Center between January 2014 and December 2019 and who accidentally ingested or aspirated dental FBs. Medical records were reviewed to determine the patients' sex, age, medical history, time from accidental ingestion of a FB to consultation, cause, location, occurrence status, nature of the FB, location of retained FB, treatment, complications, and outcome.We enrolled 32 patients (14 men, 18 women), with a mean age of 74.5 ±â€Š12.8 years. Accidental ingestion at treatment was common. The most frequent site where the FB was retained was upper gastrointestinal tract (26 cases, 81.3%). In this study, endoscopic removal was indicated for dentures under the size of 43.3 mm, for dental FB (except dentures) more than 13.6 mm. In dentures, between the number of missing teeth, clasp, type, and endoscopic removal was not statistically significant.Dentures under the size of 43.3 mm was likely to be removed by endoscope. Dental FB (except dentures) more than the size of 13.6 mm was likely to be removed by endoscope. There were no indications for endoscopic removal except for size.

Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen.

Stomata in the plant epidermis open in response to light and regulate CO2 uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H+-ATPase in guard cells. Overexpression of PM H+-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H+-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H+-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H+-ATPase in guard cells promotes the growth of perennial woody plants.

A protocol for Agrobacterium-mediated transformation of Japanese cedar, Sugi (Cryptomeria japonica D. Don) using embryogenic tissue explants.

Sugi (Cryptomeria japonica D. Don) is the most important afforestation coniferous tree in Japan. Coniferous trees normally have a long juvenile period and require a long cultivation time for breeding. Through a traditional breeding project that began in the 1950s, first generation plus trees with excellent traits were selected primarily from artificial forests and used as seedlings. Recently, the second generation plus trees obtained by crossing between plus trees have been selected. In light of this situation, the improvement of Sugi by a transgenic approach is effective in terms of shortening the breeding period compared with conventional crossing-dependent approaches. There are three key points to an efficient Agrobacterium-mediated transformation system: (1) establishment of explants with high regeneration ability, (2) optimal co-cultivation conditions for explants and Agrobacterium, and (3) efficient elimination of Agrobacterium. Here we describe a protocol for Agrobacterium-mediated transformation of Sugi that meets the above criteria using embryogenic tissues as explants isolated from immature seeds obtained by crossing.

Somatic embryogenesis in artificially pollinated seed families of 2nd generation plus trees and cryopreservation of embryogenic tissue in Cryptomeria japonica D. Don (Sugi).

Cryptomeria japonica D. Don (common name is Sugi or Japanese cedar) is the most important forestation tree species in Japan, and 2nd generation plus trees with superior traits have been selected by breeding projects. Biotechnological approaches such as genetic transformation and genome editing are expected to accelerate to add useful traits (e.g., no-pollen traits) to superior trees in short time. To develop a platform for genetic transformation and genome editing of C. japonica superior trees, this study investigated the embryogenic potential of 2nd generation plus trees and obtained good cell lines with high embryogenic potential, which could be useful material for adding new and useful traits to superior trees by genetic transformation. However, the maintenance of embryogenic cell lines is laborious, and prolonged subculture leads to a loss of embryogenesis potential. Therefore, cell lines need to be cryopreserved for long without subculture. Therefore, in this study we made a simple cryopreservation protocol suitable for most C. japonica cell lines. We showed that cryopreserved cells using this protocol formed somatic embryos, which were then converted to plantlets. Transgenic cells produced from cryopreserved cells expressed transgene, gfp. These results indicated that our cryopreservation protocol can be used for prolonged storage of genetic transformation target materials in C. japonica.

Stable expression of the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 in hybrid poplar cells.

The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 under the control of a modified cauliflower mosaic virus 35S promoter was introduced into a hybrid poplar (Populus tremula x P. tremuloides). Integration of the cbnA gene in transgenic poplar was confirmed by PCR and genomic Southern blot analysis. Expression of the cbnA gene was analyzed by Western blot analysis. Transgenic poplar calli efficiently converted 3-chlorocatechol to 2-chloro-cis,cis-muconate.

Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

Discovery of Novel Pyrido-pyridazinone Derivatives as FER Tyrosine Kinase Inhibitors with Antitumor Activity.

To obtain a new anticancer drug, we focused on FER tyrosine kinase. Starting with high-throughput screening with our in-house chemical library, compound 1, which has a pyridine moiety, was found. Referring to their X-ray crystal structure with FES proto-oncogene tyrosine kinase, as a surrogate of FER followed by chemical modification including scaffold hopping of the pyridine template, we discovered pyrido-pyridazinone derivatives with potent FER kinase inhibitory activity. Here, we disclose the structure-activity relationship on the scaffold and representative compound 21 (DS21360717), which showed in vivo antitumor efficacy in a subcutaneous tumor model.

Correlation between individual differences in intraocular pressure reduction and outflow facility due to latanoprost in normal-tension glaucoma patients.

PURPOSE: The amount of intraocular pressure (IOP) reduction achieved by the use of latanoprost eyedrops varies among patients, and there are even nonresponders. This report examines whether there is any correlation between the amount of individual variability in IOP reduction and the uveoscleral outflow facility after latanoprost eyedrop instillation in normal-tension glaucoma patients. METHODS: Sixteen normal-tension glaucoma patients (mean age, 56.4 years) were enrolled in the study to investigate the relationship between the amount of IOP reduction and outflow facility. Before treatment, subjects underwent circadian IOP measurement and then tonography, and the outflow facility was calculated. Subsequently, patients began treatment once daily with latanoprost instillation in one eye. After 4 weeks of daily latanoprost treatment, circadian IOP was measured again. RESULTS: Mean pretreatment outflow facility was 0.23 +/- 0.05 microl/min per mmHg. On average, latanoprost instillation decreased IOP by 2.8 mmHg, but the reduction varied among individuals from -0.3 mmHg to 5.8 mmHg. No significant correlation was noted between the outflow facility and the IOP decline associated with latanoprost. CONCLUSION: Because there was no significant correlation between individual IOP reduction by latanoprost and outflow facility, the differences in substantial change in uveoscleral outflow after latanoprost administration may be one explanation for the individual variation in IOP reduction after treatment with this drug.

Aqueous humor dynamics associated with the phorbol ester-induced decrease in intraocular pressure in the rabbit.

PURPOSE: To determine the effects of injection of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) into the anterior chamber of the eye on intraocular pressure (IOP) and aqueous humor dynamics. METHODS: IOP was measured for 24 h after intracameral injection of PMA (3 to 50 pmol) in unanesthetized rabbits. Aqueous humor dynamics (aqueous flow, total outflow facility, and uveoscleral outflow) were determined approximately 6 h after injection of 50 pmol of PMA in animals pretreated with indomethacin. RESULTS: Intracameral injection of 50 pmol of PMA induced a biphasic effect on IOP, consisting of a transient increase apparent at 0.5 and 1 h and a sustained decrease apparent after 2 h. This effect of PMA was dose dependent. Whereas pretreatment with indomethacin attenuated the PMA-induced increase in IOP, the sustained decrease in IOP remained apparent in the pretreated rabbits. Intracameral injection of 4alpha-PMA, an inactive PMA analog, had no effect on IOP. PMA also significantly increased uveoscleral outflow, but it had no effect on aqueous flow or total outflow facility. CONCLUSION: Intracameral injection of PMA reduced IOP in the rabbits by increasing the rate of uveoscleral outflow. This IOP-lowering effect of PMA may be mediated by activation of PKC.

Wood reinforcement of poplar by rice NAC transcription factor.

Lignocellulose, composed of cellulose, hemicellulose, and lignin, in the secondary cell wall constitutes wood and is the most abundant form of biomass on Earth. Enhancement of wood accumulation may be an effective strategy to increase biomass as well as wood strength, but currently only limited research has been undertaken. Here, we demonstrated that OsSWN1, the orthologue of the rice NAC Secondary-wall Thickening factor (NST) transcription factor, effectively enhanced secondary cell wall formation in the Arabidopsis inflorescence stem and poplar (Populus tremula×Populus tremuloides) stem when expressed by the Arabidopsis NST3 promoter. Interestingly, in transgenic Arabidopsis and poplar, ectopic secondary cell wall deposition in the pith area was observed in addition to densification of the secondary cell wall in fiber cells. The cell wall content or density of the stem increased on average by up to 38% and 39% in Arabidopsis and poplar, respectively, without causing growth inhibition. As a result, physical strength of the stem increased by up to 57% in poplar. Collectively, these data suggest that the reinforcement of wood by NST3pro:OsSWN1 is a promising strategy to enhance wood-biomass production in dicotyledonous plant species.

Ultrasound biomicroscopic classification of zonules in exfoliation syndrome.

PURPOSE: Exfoliation changes in the zonules or ciliary processes may be the earliest clinical sign of the exfoliation syndrome (XFS). In the present study we investigated the usefulness of ultrasound biomicroscopy (UBM) for detecting the early changes of XFS in zonules. METHODS: Both eyes of 11 patients with unilateral XFS and the unilateral eyes of 11 subjects without evidence of XFS were examined using UBM. The incidence of zonular changes was compared among the XFS-positive eyes of the patients, their contralateral XFS-negative eyes, and the healthy eyes of the subjects without XFS. RESULTS: Using UBM, we could successfully describe the zonules in all UBM images of all subjects. In the four quadrants, the incidence of granular-type zonules was statistically different among the three groups: it was higher in XFP-positive eyes than in XFS-negative eyes, and it was higher in XFS-negative eyes than in healthy eyes. CONCLUSIONS: UBM can detect the zonular changes that may occur early in XFS. A granular-type change in the zonules may be the most typical finding in the presence of exfoliation material. Diagnostic criteria are proposed for the early detection of XFS, based on the changes found in the zonules by UBM.