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Kaoru Araki-Sasaki,1 Yasuhiro Osakabe,2 Koji Fujita,2 Kazunori Miyata,3 Koji Hirano4 1Department of Ophthalmology, Japan Community Health Care Organization, Hoshigaoka Medical Center, Hirakata, Japan; 2Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 3Department of Ophthalmology, Miyata Eye Hospital, Miyakonojo, Japan; 4Department of Ophthalmology, Ban Buntane Hotokukai Hospital, Fujita Health University, Nagoya, Japan Abstract: We describe the histological changes in the collagen fibers of a 50-year-old male who presented keratoconus with secondary corneal amyloidosis. Corneal tissue from the patient was obtained following a penetrating keratoplasty and was subjected to histochemical analysis using Masson’s trichrome staining, Congo red staining, anti-lactoferrin antibody, and anti-transforming growth factor-beta-induced protein (TGFBIp) antibody. A Congo red-positive region was detected in the anterior half of the stroma in the center and inferior cornea. Although hemotoxylin and eosin staining revealed irregularity in the Congo red-positive region, other parts of the stroma did not show any abnormalities. Positive staining both by anti-TGFBIp and anti-lactoferrin antibodies was observed in the Congo red-positive region. Interestingly, all the layers of the corneal stroma, including the peripheral region, were positively stained by anti-TFGBIp antibody, even in the Congo red-negative area. Masson’s trichrome staining also showed irregular staining throughout the corneal stroma, even outside of the Congo red-positive region. Additionally, Bowman’s layer, which consists of collagen type IV, was damaged. TGFBIp was strongly expressed and Masson’s trichrome staining was reduced throughout the entire keratoconic stroma. The constant qualitative changes in keratoconic collagen fibers, along with the observed abnormality in the Bowman’s membrane, might point to the pathogenesis of secondary corneal amyloidosis in keratoconus. Keywords: cornea, amyloid, stroma, TGFBIp, lactoferrin

Satoko Asano, Yasuto Kunii, Hiroshi Hoshino, Yusuke Osakabe, Tetsuya Shiga, Shuntaro Itagaki, Itaru Miura, Hirooki Yabe Department of Neuropsychiatry, School of Medicine Fukushima Medical University, Fukushima, Japan Aim: Delirium is commonly encountered in daily clinical practice. To identify predictors influencing outcomes, we retrospectively examined the characteristics of inpatients with delirium who required psychiatric medication during hospitalization.Methods: We extracted all new inpatients (n=523) consulted for psychiatric symptoms at Fukushima Medical University Hospital between October 2011 and September 2013. We selected 203 inpatients with delirium diagnosed by psychiatrists. We analyzed data from 177 inpatients with delirium who received psychiatric medication. We defined an “early improvement group” in which delirium resolved in ≤3 days after starting psychiatric medication, and a “prolonged group” with delirium lasting for >3 days. Among the 83 inpatients with renal dysfunction (estimated glomerular filtration rate 3 days. We then examined differences between groups for different categorical variables.Results: Dose of antipsychotic medication at end point was significantly lower in the prolonged group with renal dysfunction than in the early improvement group with renal dysfunction.Conclusion: The results suggest that maintaining a sufficient dose of antipsychotics from an early stage may prevent prolongation of delirium even in inpatients with renal dysfunction. Keywords: antipsychotic, prolonged delirium, chronic kidney disease, pharmacokinetics

Koji Hirano,1 Mihoko Koide,2 Yoshikazu Mizoguchi,3 Yasuhiro Osakabe,4 Kaoru-Araki Sasaki5 1Department of Ophthalmology, Ban Buntane Hotokukai Hospital, School of Medicine, Fujita Health University, Nagoya, Japan; 2Koide Internal Medicine and Eye Clinic, Nagoya, Japan; 3Department of Pathology, Ban Buntane Hotokukai Hospital, School of Medicine, Fujita Health University, Nagoya, Japan; 4Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 5Department of Ophthalmology, Japan Health Care Organization, Hoshigaoka Medical Center, Hirakata, Japan Purpose: Leukoplakia is the term given to a white patch or plaque that is found mainly on the oral mucus membrane. It can occasionally be seen on the corneal surface. We report our clinical and histopathological findings in a case of unilateral corneal leukoplakia. Methods: A 26-year-old woman was referred to our hospital because of a white patch on her right cornea that continued to expand. She first noticed the white patch when she was 20 years old, and the white patch had expanded to cover the pupillary area affecting her vision. After plastic surgery on both eyelids for bilateral entropion to alleviate the pain caused by the eyelashes rubbing the cornea, the white corneal patch decreased in size. Because of this reduction, we performed surgery to remove the patch with microforceps under topical anesthesia. The plaque was removed easily and completely, and submitted for histopathological examination. Results: Histopathological examination showed that the specimen had characteristics of epidermis with a basal cell layer, spinous cell layer, granular cell layer, and horny layer with hyperkeratosis. She was diagnosed with leukoplakia of the corneal surface. The basic structure of the squamous cell layer was preserved, and there were no signs of metaplasia. Six months after the removal of the leukoplakia, no recurrence was seen and her corrected decimal visual acuity recovered to 1.0. Conclusion: Our case of unilateral corneal leukoplakia without limbal involvement was most likely caused by chronic irritation of the cornea by the eyelashes. Although it was totally removed with good recovery of vision, we continue to follow the patient because of the potential of malignant transformation. Keywords: corneal leukoplakia, hyperkeratosis, eyelashes, chronic friction

Kaoru Araki-Sasaki,1 Yasuhiro Osakabe,2 Hideki Fukuoka,3 Ryuichi Ideta,4 Koji Hirano5 1Department of Ophthalmology, Japan Community Health Care Organization, Hoshigaoka Medical Center, Hirakata, 2Department of Molecular Pathology, Tokyo Medical University, Tokyo, 3Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, 4Ideta Eye Hospital, Kumamoto, 5Department of Ophthalmology, Ban Buntane Hotokukai Hospital, School of Medicine, Fujita Health University, Nagoya, Japan Purpose: To describe observations by ultrahigh-resolution optical coherence tomography (OCT) in a secondary corneal amyloidosis (SCA) patient with histological analysis of excised tissue. A unique finding under OCT of her fellow eye is also described.Case: A 39-year-old female had suffered from trichiasis in both of her eyes for more than 30 years. Slit-lamp examination showed a milky-white soft mass on her left cornea and a linear opacity on the fellow cornea at the cilia-attached region. OCT demonstrated the presence of a mass region within a thin epithelial layer and no destruction of Bowman’s layer in her left cornea. In the fellow cornea, which exhibited a linear opacity, a high-density spot in Bowman’s layer was observed at the cilia-attached region covered by the epithelial layer, with normal thickness. Histological examination of the excised cornea showed that the mass was positive with both Congo red and antilactoferrin antibody.Conclusion: SCA, amyloid gradually accumulates above Bowman’s layer, occupying the epithelial layer, with no destruction of Bowman’s layer until the advanced stage. A high-density spot in Bowman’s layer might be the first stage of SCA. Keywords: OCT, secondary corneal amyloidosis, lactoferrin, cornea

Kaoru Araki-Sasaki,1,2 Atsuko Fukumoto,1 Yasuhiro Osakabe,3 Hideya Kimura,1 Shinichiro Kuroda1 1Nagata Eye Clinic, Nara, Japan; 2Department of Ophthalmology, Japan Community Health Care Organization, Hoshigaoka Medical Center, Osaka, Japan; 3Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan Abstract: The purpose of this study was to describe the clinical characteristics of fungal keratitis caused by Candida albicans in an eye after Descemet stripping automated endothelial keratoplasty (DSAEK). A 72-year-old male with a history of three trabeculectomies, cataract surgery, and two DSAEK procedures developed a corneal ulcer in his right eye two years after his last DSAEK. Fungal keratitis was most likely related to the immunosuppressive conditions that occurred due to the previous operations, the continuous use of steroid eye drops, and the use of disposable soft contact lenses. A smear and culture from the ulcer detected Candida albicans. Slit-lamp examination showed the characteristic feature was the presence of interface infiltrates located between the host and the graft cornea and in the enlarged area around the ulcer. Two weeks after intense antimycotic treatments with voriconazole, miconazole, and natamycin, perforation of the cornea occurred and further therapeutic penetrating keratoplasty was required. Histological analysis revealed an accumulation of infiltrated cells and fibrotic tissue. The poor prognosis for fungal keratitis that occurs in eyes after undergoing DSAEK may be related to the rapid expansion of inflammatory cells through the interface between the host and the graft. In eyes that develop fungal keratitis after DSAEK, special attention should be paid to the possibility that perforation could occur in these patients. Keywords: DSAEK, Candida albicans, fungal keratitis, keratomycosis, post-operative infection

An association between increased anti-SARS-CoV-2 spike IgA after COVID-19 vaccination and reactivation of pre-existing IgA vasculitis has also been reported.[5] Renal pathological findings in our case included prominent mesangial proliferative changes, intracapillary proliferation, glomerular necrosis/rupture and crescent formation. Learning points for clinicians The coronavirus disease 2019 (COVID-19) mRNA vaccine can cause flare ups of glomerulonephritis, especially in patients with IgA vasculitis. [Extracted from the article]

In grass cell walls, ferulic acid (FA) serves as an important cross-linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR-Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX-bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH-edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH-edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall-bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall-bound p-coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ-oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH-edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross-linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Purpose: To clarify the clinical and pathologic findings of 7 patients with inflamed juvenile conjunctival nevus (IJCN) treated with tacrolimus., Study Design: Retrospective study., Subjects and Methods: The medical records of 7 male patients diagnosed with IJCN between February 2007 and October 2022 at the Kansai Medical University Hospital and Ideta Eye Hospital were retrospectively reviewed. The patients' mean age was 11.3 (8-20) years, and the average follow-up period was 18 months. The patients underwent anterior segment optical coherence tomography (AS-OCT) and anterior segment optical coherence tomography angiography (AS-OCTA). All the patients were treated with steroids or immunosuppressant eye drops, or both. Histopathologic examinations were performed on specimens resected from 2 patients., Results: The clinical characteristics of IJCN were as follows: (1) presence of cysts within a pigmented conjunctival mass, (2) tumor accompanied by feeder vessels, and (3) predominantly found in boys with allergic diseases. AS-OCT and AS-OCTA revealed the presence of a cystic structure with poor vascular signals within it and numerous vessels distributed on the sclera underlying the lesion. Immunosuppressant eye drops relieved the congestion, diminished the inflow vessels, and induced shrinkage of the cysts in all the patients. Histopathologic examinations revealed infiltration of inflammatory cells such as lymphocytes and macrophages and low proliferative activity of melanocytes with no division pattern., Conclusions: This study clarified the clinical and histologic characteristics of IJCN. Noninvasive AS-OCT and AS-OCTA might be useful for diagnosing IJCN. The histopathologic findings of this study indicate that immunosuppressive and antiallergic agents can effectively bring about disease remission., Competing Interests: Declarations Conflict of interest A. Tomoda, None; K. Araki-Sasaki, None; H. Obata, None; S. Ideta, None; M. Kuroda, None; K. Fujita, None; Y. Osakabe, None; K. Takahashi, None., (© 2024. The Author(s).)

Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involve insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, using such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes successfully developed and were regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Sulfur (S) is an essential macronutrient for plant growth and metabolism. SULTR2;1 is a low-affinity sulfate transporter facilitating the long-distance transport of sulfate in Arabidopsis. The physiological function of SULTR2;1 in the plant life cycle still needs to be determined. Therefore, we analyzed the sulfate transport, S-containing metabolite accumulation and plant growth using Arabidopsis SULTR2;1 disruption lines, sultr2;1-1 and sultr2;1-2, from seedling to mature growth stages to clarify the metabolic and physiological roles of SULTR2;1. We observed that sulfate distribution to the stems was affected in sultr2;1 mutants, resulting in decreased levels of sulfate, cysteine, glutathione (GSH) and total S in the stems, flowers and siliques; however, the GSH levels increased in the rosette leaves. This suggested the essential role of SULTR2;1 in sulfate transport from rosette leaves to the primary stem. In addition, sultr2;1 mutants unexpectedly bolted earlier than the wild-type without affecting the plant biomass. Correlation between GSH levels in rosette leaves and the bolting timing suggested that the rosette leaf GSH levels or limited sulfate transport to the early stem can trigger bolting. Overall, this study demonstrated the critical roles of SULTR2;1 in maintaining the S metabolite levels in the aerial part and transitioning from the vegetative to the reproductive growth phase., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Background: Amphiregulin (AREG) is a ligand of epidermal growth factor receptor (EGFR), which plays an important role in injury-induced kidney fibrosis. However, the clinical significance of serum soluble AREG in chronic kidney disease (CKD) is unclear. In this study, we elucidated the clinical significance of serum soluble AREG in CKD by analyzing the association of serum soluble AREG levels with renal function and other clinical parameters in patients with CKD., Methods: In total, 418 Japanese patients with CKD were enrolled, and serum samples were collected for the determination of soluble AREG and creatinine (Cr) levels, and other clinical parameters. Additionally, these parameters were evaluated after 2 and 3 years. Moreover, immunohistochemical assay was performed ate AREG expression in the kidney tissues of patients with CKD., Results: Soluble AREG levels were positively correlated with serum Cr (p < 0.0001). Notably, initial AREG levels were positively correlated with changes in renal function (ΔCr) after 2 (p < 0.0001) and 3 years (P = 0.048). Additionally, soluble AREG levels were significantly higher (p < 0.05) in patients with diabetic nephropathy or primary hypertension. Moreover, AREG was highly expressed in renal tubular cells in patients with advanced CKD, but only weakly expressed in patients with preserved renal function., Conclusion: Serum soluble AREG levels were significantly correlated with renal function, and changes in renal function after 2 and 3 years, indicating that serum soluble AREG levels might serve as a biomarker of renal function and renal prognosis in CKD., (© 2024. The Author(s), under exclusive licence to Japanese Society of Nephrology.)

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants' cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Speech-sound stimuli have a complex structure, and it is unclear how the brain processes them. An event-related potential (ERP), known as mismatch negativity (MMN), is elicited when an individual's brain detects a rare sound. In this study, MMNs were measured in response to an omitted segment of a complex sound consisting of a Japanese vowel. The results indicated that the latency from onset in the right hemisphere was significantly shorter than that in the frontal midline and left hemispheres during left ear stimulation. Additionally, the results of latency from omission showed that the latency of stimuli omitted in the latter part of the temporal window of integration (TWI) was longer than that of stimuli omitted in the first part of the TWI. The mean peak amplitude was found to be higher in the right hemisphere than in the frontal midline and left hemispheres in response to left ear stimulation. In conclusion, the results of this study suggest that would be incorrect to believe that the stimuli have strictly the characteristics of speech-sound. However. the results of the interaction effect in the latencies from omission were insignificant. These results suggest that the detection time for deviance may not be related to the stimulus ear. However, the type of deviant stimuli on latencies was found to be significant. This is because the detection of the deviants was delayed when a deviation occurred in the latter part of the TWI, regardless of the stimulation of the ear., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Bioengineering approaches to modify lignin content and structure in plant cell walls have shown promise for facilitating biochemical conversions of lignocellulosic biomass into valuable chemicals. Despite numerous research efforts, however, the effect of altered lignin chemistry on the supramolecular assembly of lignocellulose and consequently its deconstruction in lignin-modified transgenic and mutant plants is not fully understood. In this study, we aimed to close this gap by analyzing lignin-modified rice (Oryza sativa L.) mutants deficient in 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (CAldOMT) and CINNAMYL ALCOHOL DEHYDROGENASE (CAD). A set of rice mutants harboring knockout mutations in either or both OsCAldOMT1 and OsCAD2 was generated in part by genome editing and subjected to comparative cell wall chemical and supramolecular structure analyses. In line with the proposed functions of CAldOMT and CAD in grass lignin biosynthesis, OsCAldOMT1-deficient mutant lines produced altered lignins depleted of syringyl and tricin units and incorporating noncanonical 5-hydroxyguaiacyl units, whereas OsCAD2-deficient mutant lines produced lignins incorporating noncanonical hydroxycinnamaldehyde-derived units. All tested OsCAldOMT1- and OsCAD2-deficient mutants, especially OsCAldOMT1-deficient lines, displayed enhanced cell wall saccharification efficiency. Solid-state nuclear magnetic resonance (NMR) and X-ray diffraction analyses of rice cell walls revealed that both OsCAldOMT1- and OsCAD2 deficiencies contributed to the disruptions of the cellulose crystalline network. Further, OsCAldOMT1 deficiency contributed to the increase of the cellulose molecular mobility more prominently than OsCAD2 deficiency, resulting in apparently more loosened lignocellulose molecular assembly. Such alterations in cell wall chemical and supramolecular structures may in part account for the variations of saccharification performance of the OsCAldOMT1- and OsCAD2-deficient rice mutants., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Genome editing has revolutionized plant research and plant breeding by enabling precise genome manipulation. In particular, the application of type II CRISPR-Cas9 systems to genome editing has proved an important milestone, accelerating genetic engineering and the analysis of gene function. On the other hand, the potential of other types of CRISPR-Cas systems, especially many of the most abundant type I CRISPR-Cas systems, remains unexplored. We recently developed a novel genome editing tool, TiD, based on the type I-D CRISPR-Cas system. In this chapter, we describe a protocol for genome editing of plant cells using TiD. This protocol allows the application of TiD to induce short insertion and deletions (indels) or long-range deletions at target sites with high specificity in tomato cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The 4-coumarate:coenzyme A ligase (4CL) is a key enzyme that contributes to channeling metabolic flux in the cinnamate/monolignol pathway, leading to the production of monolignols, p-hydroxycinnamates, and a flavonoid tricin, the major building blocks of lignin polymer in grass cell walls. Vascular plants often contain multiple 4CL genes; however, the contribution of each 4CL isoform to lignin biosynthesis remains unclear, especially in grasses. In this study, we characterized the functions of two rice (Oryza sativa L.) 4CL isoforms (Os4CL3 and Os4CL4) primarily by analyzing the cell wall chemical structures of rice mutants generated by CRISPR/Cas9-mediated targeted mutagenesis. A series of chemical and nuclear magnetic resonance analyses revealed that loss-of-function of Os4CL3 and Os4CL4 differently altered the composition of lignin polymer units. Loss of function of Os4CL3 induced marked reductions in the major guaiacyl and syringyl lignin units derived from both the conserved non-γ-p-coumaroylated and the grass-specific γ-p-coumaroylated monolignols, with more prominent reductions in guaiacyl units than in syringyl units. In contrast, the loss-of-function mutation to Os4CL4 primarily decreased the abundance of the non-γ-p-coumaroylated guaiacyl units. Loss-of-function of Os4CL4, but not of Os4CL3, reduced the grass-specific lignin-bound tricin units, indicating that Os4CL4 plays a key role not only in monolignol biosynthesis but also in the biosynthesis of tricin used for lignification. Further, the loss-of-function of Os4CL3 and Os4CL4 notably reduced cell-wall-bound ferulates, indicating their roles in cell wall feruloylation. Overall, this study demonstrates the overlapping but divergent roles of 4CL isoforms during the coordinated production of various lignin monomers., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cytokinin plays an important role in plant stress responses via a multistep signaling pathway, involving the histidine phosphotransfer proteins (HPs). In Arabidopsis thaliana, the AHP2, AHP3 and AHP5 proteins are known to affect drought responses; however, the role of AHP4 in drought adaptation remains undetermined. In the present study, using a loss-of-function approach we showed that AHP4 possesses an important role in the response of Arabidopsis to drought. This is evidenced by the higher survival rates of ahp4 than wild-type (WT) plants under drought conditions, which is accompanied by the downregulated AHP4 expression in WT during periods of dehydration. Comparative transcriptome analysis of ahp4 and WT plants revealed AHP4-mediated expression of several dehydration- and/or abscisic acid-responsive genes involved in modulation of various physiological and biochemical processes important for plant drought acclimation. In comparison with WT, ahp4 plants showed increased wax crystal accumulation in stems, thicker cuticles in leaves, greater sensitivity to exogenous abscisic acid at germination, narrow stomatal apertures, heightened leaf temperatures during dehydration, and longer root length under osmotic stress. In addition, ahp4 plants showed greater photosynthetic efficiency, lower levels of reactive oxygen species, reduced electrolyte leakage and lipid peroxidation, and increased anthocyanin contents under drought, when compared with WT. These differences displayed in ahp4 plants are likely due to upregulation of genes that encode enzymes involved in reactive oxygen species scavenging and non-enzymatic antioxidant metabolism. Overall, our findings suggest that AHP4 plays a crucial role in plant drought adaptation., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: In Japan, only two medications of immediate-release levodopa with distinct ratios of decarboxylase inhibitor (DCI), namely levodopa/benserazide 100/25 mg and levodopa/carbidopa 100/10 mg, are available for the treatment of Parkinson's disease (PD). The relationship between the difference in the DCI to levodopa ratio and the development of motor complications in long-term administration of levodopa is unknown., Purpose: We assessed the duration from initiation of levodopa/DCI to the emergence of motor fluctuations in patients with PD treated with levodopa/benserazide and levodopa/carbidopa., Methods: We retrospectively assessed the disease course, especially the period from the onset of motor symptoms or initiation of levodopa/DCI to the emergence of motor fluctuations, in patients with PD who were initially treated with either levodopa/benserazide (300/75 mg/day) or levodopa/carbidopa (300/30 mg/day)., Results: Of the 186 candidates, 52 patients were enrolled. The mean duration to the emergence of motor fluctuations in the levodopa/carbidopa group was significantly longer than that in the levodopa/benserazide group (5.0 ± 1.4 vs 3.1 ± 1.2 years, p < 0.01). The mean duration from onset of motor symptoms to the emergence of motor fluctuations in the levodopa/carbidopa group was also significantly longer than that in the levodopa/benserazide group (6.6 ± 1.6 vs 4.7 ± 1.3 years, p < 0.01)., Conclusion: Our study suggests that levodopa/carbidopa therapy with a DCI to levodopa ratio of 1:10 may delay the occurrence of motor fluctuations when compared to levodopa/benserazide therapy with that of 1:4. The difference in the blending ratio of levodopa/DCI may influence the disease progression in PD., (Copyright © 2021. Published by Elsevier B.V.)

Since its first appearance, CRISPR-Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR-Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR-Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR-Cas systems have been reported. Among them, several CRISPR-Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR-Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR-Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR-Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR-Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR-Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR-Cas tools are helping expand our plant genome engineering toolbox., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Background: The interferon (IFN)-γ release assay (IGRA) has recently been established as a method to evaluate the infection status of tuberculosis instead of the tuberculin skin test. However, indeterminate results can create challenges to interpretation. The IGRA has been available in Japan since 2005, including the recently launched QuantiFERON-TB Gold Plus (QFT-plus) assay., Objectives: The aim of this study was to investigate the clinical features and predictors of indeterminate results by the QFT-plus test in routine practice., Methods: This was a cross-sectional study of 1258 patients. Multivariate logistic regression models were employed to investigate the clinical factors related to indeterminate results by the QFT-plus., Results: Overall, 91.8% of results were found to be conclusive and 8.2% were indeterminate. The QFT-plus indeterminate results were predominantly due to a low level of IFN-γ production by mitogens. Multivariate analysis indicated that an indeterminate result was significantly associated with age, sex, corticosteroid use, autoimmune disease, and inpatient setting., Conclusion: Certain types of individuals are at higher risk of an indeterminate IGRA result. The QFT-plus test for hospitalized patients should be avoided as much as possible, and it is better to perform the test for those patients in outpatient settings.

Background: Cognitive decline after oral administration of sedatives, such as benzodiazepines, is a serious side effect. Suvorexant, an orexin receptor antagonist, has a favorable tolerability and a limited side-effect profile., Aim: The purpose of this study was to estimate the cognitive decline 1 day after oral medication with lormetazepam, a benzodiazepine, and suvorexant by comparing mismatch negativity (MMN) and P300 reflecting auditory discrimination function., Methods: Sixty healthy subjects (42 males) were randomly assigned to three groups receiving suvorexant 20 mg, lormetazepam 2 mg, or placebo in this double-blind, randomized control study. Event-related potential recordings during an auditory oddball task and a digit symbol substitution test (DSST) were performed 1 day after oral administration., Results: MMN, on the day after oral administration, was significantly attenuated in the lormetazepam group compared with the other two groups, but there was no difference between the suvorexant and placebo groups. No significant difference was found in P300 amplitudes and DSST scores among the three groups., Conclusion: These findings suggest that suvorexant, unlike benzodiazepine, is not associated with cognitive deficits, as revealed by MMN but not P300. This study shows a neurophysiological difference in the effects of suvorexant and benzodiazepine on cognitive function.

Rationale: As a treatment for cognitive dysfunction in schizophrenia, oxytocin nasal sprays potentially improve social cognition, facial expression recognition, and sense of smell. Mismatch negativity (MMN) is an event-related potential (ERP) reflecting auditory discrimination while MMN deficits reflect cognitive function decline in schizophrenia., Objectives: To determine whether oxytocin nasal spray affects auditory MMN METHODS: We measured ERPs in healthy subjects during an auditory oddball task, both before and after oxytocin nasal spray administration. Forty healthy subjects were randomly assigned to either the oxytocin or placebo group. ERPs were recorded during the oddball task for all subjects before and after a 24 international unit (IU) intranasal administration, and MMN was compared between the two groups., Results: Participants who received oxytocin had significantly shorter MMN latencies than those who received a placebo. Oxytocin had no significant effect on the Change in MMN amplitude., Conclusions: The shortened MMN latencies that were observed after oxytocin nasal spray administration suggest that oxytocin may promote the comparison-decision stage.

Adoption of CRISPR-Cas systems, such as CRISPR-Cas9 and CRISPR-Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I-the most abundant CRISPR system in bacteria-remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense ('Cascade': Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR-Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR-Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR-Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tomato INDOLE-3-ACETIC ACID9 ( SlIAA9 ) is a transcriptional repressor in auxin signal transduction, and SlIAA9 knockout tomato plants develop parthenocarpic fruits without fertilization. We generated sliaa9 mutants with parthenocarpy in several commercial tomato cultivars (Moneymaker, Rio Grande, and Ailsa Craig) using CRISPR-Cas9, and null-segregant lines in the T1 generation were isolated by self-pollination, which was confirmed by PCR and Southern blot analysis. We then estimated shoot growth phenotypes of the mutant plants under different light (low and normal) conditions. The shoot length of sliaa9 plants in Moneymaker and Rio Grande was smaller than those of wild-type cultivars in low light conditions, whereas there was not clear difference between the mutant of Ailsa Craig and the wild-type under both light conditions. Furthermore, young seedlings in Rio Grande exhibited shade avoidance response in hypocotyl growth, in which the hypocotyl lengths were increased in low light conditions, and sliaa9 mutant seedlings of Ailsa Craig exhibited enhanced responses in this phenotype. Fruit production and growth rates were similar among the sliaa9 mutant tomato cultivars. These results suggest that control mechanisms involved in the interaction of AUX/IAA9 and lights condition in elongation growth differ among commercial tomato cultivars., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abe-Hara, Yamada, Wada, Ueta, Hashimoto, Osakabe and Osakabe.)

Objective: We hypothesized that sensory memory associated with the temporal window of integration (TWI) would be impaired in patients with schizophrenia, an issue that had not been evaluated using omission mismatch negativity (MMN) of complex speech sounds. We aimed to assess the functional changes in auditory sensory memory associated with the TWI in patients with schizophrenia by investigating the effect of omission of complex speech stimuli on the MMN., Methods: In total, 17 patients with schizophrenia and 15 control individuals participated in the study. The MMN in response to omission deviants of complex speech sounds was recorded, while the participants were instructed to ignore the series of speech sounds., Results: The MMN latency in patients with schizophrenia was significantly prolonged by deviant stimuli to omissions corresponding to the early and late parts of the temporal TWI. There were no significant group differences in the amplitude of the MMN to omissions at different time points across the TWI., Conclusions: Our results suggested that sensory tracing function in patients with schizophrenia is impaired in the early and the later half of the TWI., Significance: We showed that certain MMN abnormalities in patients with schizophrenia may be caused by an impaired TWI., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

A 55-year-old woman with neuromyelitis optica (NMO) had recurrent myalgias with hyperCKemia. A muscle biopsy suggested nonspecific myopathic changes. Regarding immunohistochemistry, the expression of both major histocompatibility complex class I and myxovirus resistance protein A was observed in the endomysial capillaries, suggesting immunological involvement of these capillaries, whereas both C5b9 (membrane attack complex) and aquaporin 4 immunofluorescence stainings were normal. The present findings led us to conclude that one possible mechanism for hyperCKemia in NMO underlying the immunological involvement of the endomysial capillaries was an as-yet-unidentified factor that triggered damage to the integrity of the sarcolemma and thereby cause CK leakage into the serum.

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I-the most abundant CRISPR system in bacteria-has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

Background: Bronchoscopic examinations are vital to diagnose pulmonary diseases. However, as coughing is triggered during and after the procedure, it is imperative to take measures against nosocomial infections, especially for airborne infections like tuberculosis (TB). The interferon-γ release assay (IGRA) has recently been established as a method to evaluate the infection status of TB. We aimed to ascertain the efficacy of IGRA and clinical findings in estimating the prevalence of active TB before bronchoscopy., Methods: We obtained IGRA results from 136 inpatients using a QuantiFERON-TB Gold In-Tube test. Bronchoscopy samples were cultured in Mycobacteria Growth indicator tubes and 2% Ogawa solid medium. We evaluated the adjusted effects of multiple clinical variables on active TB status using a logistic regression model. In addition, multiple variables were converted into a decision tree to predict active TB., Results: Five (3.7%) patients were diagnosed with culture-positive TB, two of whom were simultaneously diagnosed with non-small-cell lung carcinoma or small-cell lung carcinoma. The multivariate analysis suggested the probability of predicting active TB using the IGRA [odds ratio (OR), 72.7; 95% confidence interval (CI), 3.169-1668; P=0.007] and decreased estimated glomerular filtration rate (eGFR) (OR, 0.937; 95% CI, 0.882-0.996; P=0.038) in patients undergoing bronchoscopy. A decision tree validated the use of these two variables to predict active TB., Conclusions: IGRA test results are useful for predicting active TB before bronchoscopy. This strategy could identify patients who require antibiotic therapy to prevent TB or who are in the active phase of TB., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jtd-19-3653). The authors have no conflicts of interest to declare., (2020 Journal of Thoracic Disease. All rights reserved.)