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Background: Osteoarthritis of the thumb carpometacarpal joint often presents pain and affects activity of daily life. There is no consensus regarding prevalence of primary radiographic osteoarthritis and symptomatic osteoarthritis of the thumb carpometacarpal joint, and their associated factors are not well investigated. The purpose of this study is to evaluate the prevalence and associated factors of primary radiographic and symptomatic osteoarthritis of the thumb carpometacarpal joint in a resident cohort., Methods: A total of 1297 residents aged 50 to 89 were randomly sampled from a small rural town in Japan, and their demographic data and questionnaire regarding lifestyle and job were obtained. All participants underwent posteroanterior radiographs of the bilateral hands. Participants with thumb carpometacarpal joint radiographic osteoarthritis were diagnosed if one of their thumb carpometacarpal joints was Kellgren-Lawrence grade 2, 3, or 4. Symptomatic osteoarthritis was diagnosed by tenderness or positive motion pain at the thumb base. Associated factors for the thumb carpometacarpal joint radiographic osteoarthritis were statistically analyzed., Results: A total of 646 thumbs with 323 participants (153 male and 170 female) were investigated. The mean age was 69.1 (range 50-89) years, The prevalence of thumb carpometacarpal joint radiographic osteoarthritis was 17.3 % (13.7 % males and 20.6 % females) and that of thumb carpometacarpal joint symptomatic osteoarthritis was 3.1 %. Associated factors for thumb carpometacarpal joint radiographic osteoarthritis were presence of finger joint radiographic osteoarthritis in the ipsilateral hand, increasing age, and female in order of degree of relevance. Dominant hand, body mass index, smoking history, heavy manual or agriculture work, use of vibrating tools, racket or bat sports, or scaphotapeziotrapezoidal joint radiographic osteoarthritis were not associated factors for thumb carpometacarpal joint radiographic osteoarthritis., Conclusions: The prevalence of thumb carpometacarpal joint primary radiographic osteoarthritis in general Japanese elderly aged 50-89 years was 17.3 %, most of carpometacarpal joint radiographic osteoarthritis was asymptomatic., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Competing Interests: The authors have no conflicts of interest to disclose.

The NPR proteins function as salicylic acid (SA) receptors in Arabidopsis thaliana. AtNPR1 plays a central role in SA-induced transcriptional reprogramming whereby positively regulates SA-mediated defense. NPRs are found in the genomes of nearly all land plants. However, we know little about the molecular functions and physiological roles of NPRs in most plant species. We conducted phylogenetic and alignment analyses of NPRs from 68 species covering the significant lineages of land plants. To investigate NPR functions in bryophyte lineages, we generated and characterized NPR loss-of-function mutants in the liverwort Marchantia polymorpha. Brassicaceae NPR1-like proteins have characteristically gained or lost functional residues identified in AtNPRs, pointing to the possibility of a unique evolutionary trajectory for the Brassicaceae NPR1-like proteins. We find that the only NPR in M. polymorpha, MpNPR, is not the master regulator of SA-induced transcriptional reprogramming and negatively regulates bacterial resistance in this species. The Mpnpr transcriptome suggested roles of MpNPR in heat and far-red light responses. We identify both Mpnpr and Atnpr1-1 display enhanced thermomorphogenesis. Interspecies complementation analysis indicated that the molecular properties of AtNPR1 and MpNPR are partially conserved. We further show that MpNPR has SA-binding activity. NPRs and NPR-associated pathways have evolved distinctively in diverged land plant lineages to cope with different terrestrial environments., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Background: Insertion of electrode catheters into the coronary sinus (CS) through the right internal jugular vein (RIJV) carries risks of pneumothorax and severe hematoma formation. This study was performed to compare the safety and feasibility of catheterization through the left cubital superficial vein versus the RIJV., Methods: This prospective nonrandomized study involved consecutive patients who underwent catheter ablation from September 2021 to February 2023. Blind puncture techniques were used in the left cubital vein group; ultrasound-guided insertion was performed in the RIJV group. The success rates of sheath insertion and CS catheterization, the procedure and fluoroscopy times of CS cannulation, and complications were compared between groups., Results: The left cubital vein group comprised 152 patients, and the RIJV group comprised 58 patients. The sheath insertion success rate was significantly lower in the cubital vein group than in the RIJV group (84.9% vs 100%, respectively; p  = .0008). In the cubital vein group, blind puncture attempts failed in 20 patients; three patients developed guidewire-induced venous injury. One arterial puncture occurred in the RIJV group. After successful sheath insertion, no significant differences were observed in the CS cannulation success rate (97% vs 100%, p  = .55), procedure time (median [range], 93 [51-174] vs 74 [44-129] s; p  = .19), or fluoroscopy time (median [range], 66 [36-134] vs 48 [30-92] s; p  = .17). No serious complications requiring procedural discontinuation occurred., Conclusion: The left cubital vein approach is practical, offering a viable alternative to the RIJV approach., Competing Interests: Authors declare no conflict of interests for this article., (© 2024 The Author(s). Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of Japanese Heart Rhythm Society.)

Background: We explore an optimized approach for increasing lesion size using a novel ablation catheter with a surface thermocouple and efficient irrigation in a temperature-control setting., Methods: We conducted radiofrequency applications at various power levels (35 W, 40 W, and 45 W), contact forces (CFs, 10 g/20 g), and durations (60 s/120 s/180 s) in perpendicular/parallel catheter orientations, with normal saline irrigation (NS-irrigation) and Half NS-irrigation (HNS-irrigation) in an ex-vivo model (Step 1). In addition, we performed applications (35 W/40 W/45 W for 60 s/120 s/180 s in NS-irrigation and 35 W/40 W for 60 s/120 s/180 s in HNS-irrigation) in four swine (Step 2), evaluating lesion characteristics and the occurrence of steam pops., Results: In Step 1, out of 288 lesions, we observed 47 (16.3%) steam pops, with 13 in NS-irrigation and 34 in HNS-irrigation ( p  = .001). Although steam pops were mostly observed with the most aggressive setting (45 W/180 s, 54%) with NS-irrigation, they happened in less aggressive settings with HNS irrigation. Lesion size significantly increased with longer-duration ablation but not with HNS-irrigation. The optimal %impedance-drop cutoff to predict steam pops was 20% with a negative-predictive-value (NPV) = 95.1% including NS- and HNS-irrigation groups, and 22% with an NPV = 96.1% in NS-irrigation group. In Step 2, similar to the ex-vivo model, lesion size significantly increased with longer-duration ablation but not with HNS-irrigation. Steam pops were absent with NS-irrigation (0/35) even with the largest %impedance-drop reaching 31% at 45 W/180 s. All steam pops were observed with HNS-irrigation (6/21, 29%). The optimal %impedance-drop cutoff predicting steam pops was 24% with an NPV = 96.3% including both NS- and HNS-irrigation groups., Conclusions: Rather than using HNS-irrigation, very long-duration of radiofrequency applications up to 45 W/180 s may be recommended to safely and effectively increase lesion dimensions using this catheter with NS-irrigation., Competing Interests: Drs. Goto, Takigawa, and Miyazaki belong to the division which receives research endowments from Medtronic Japan, Boston Scientific, Japan Lifeline, APEX, and WIN international. Dr. Takigawa received a lecture fee from Abbott Medical Japan, LIS. Dr. Ohuchi received a joint research fund from Abbott Medical Japan, LIS. No other authors have conflict of interest to declare., (© 2024 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of Japanese Heart Rhythm Society.)

Although it is well known that hierarchical transcriptional networks are essential for various aspects of plant development and environmental response, little has been investigated about whether and how they also regulate the plant cell cycle. Recent studies on cell cycle regulation in Arabidopsis thaliana identified SCARECROW-LIKE28 (SCL28), a GRAS-type transcription factor, that constitutes a hierarchical transcriptional pathway comprised of MYB3R, SCL28 and SIAMESE-RELATED (SMR). In this pathway, MYB3R family proteins regulate the G2/M-specific transcription of the SCL28 gene, of which products, in turn, positively regulate the transcription of SMR genes encoding a group of plant-specific inhibitor proteins of cyclin-dependent kinases. However, this pathway with a role in cell cycle inhibition is solely demonstrated in A. thaliana , thus leaving open the question of whether and to what extent this pathway is evolutionarily conserved in plants. In this study, we conducted differential display RT-PCR on synchronized Nicotiana tabacum (tobacco) BY-2 cells and identified several M-phase-specific cDNA clones, one of which turned out to be a tobacco ortholog of SCL28 and was designated NtSCL28 . We showed that NtSCL28 is expressed specifically during G2/M and early G1 in the synchronized cultures of BY-2 cells. NtSCL28 contains MYB3R-binding promoter elements, so-called mitosis-specific activator elements, and is upregulated by a hyperactive form of NtmybA2, one of the MYB3R proteins from tobacco. Our study indicated that a part of the hierarchical pathway identified in A. thaliana is equally operating in tobacco cells, suggesting the conservation of this pathway across different families in evolution of angiosperm., Competing Interests: Conflict of interestThe authors declare no conflict of interest., (© 2023 Japanese Society for Plant Biotechnology.)

Background: This study aimed to establish a systematic method for diagnosing atrioventricular nodal reentrant tachycardia (AVNRT) with a bystander concealed nodoventricular pathway (cNVP)., Methods: We analyzed 13 cases of AVNRT with a bystander cNVP, 11 connected to the slow pathway (cNVP-SP) and two to the fast pathway (cNVP-FP), along with two cases of cNVP-related orthodromic reciprocating tachycardia (ORT)., Results: The diagnostic process was summarized in three steps. Step 1 was identification of the presence of an accessory pathway by resetting the tachycardia with delay (n = 9) and termination without atrial capture ( n  = 4) immediately after delivery of a His-refractory premature ventricular contraction (PVC). Step 2 was exclusion of ORT by atrio-His block during the tachycardia ( n  = 4), disappearance of the reset phenomenon after the early PVC ( n  = 7), or dissociation of His from the tachycardia during ventricular overdrive pacing (n = 1). Moreover, tachycardia reset/termination without the atrial capture ( n  = 2/2) 1 cycle after the His-refractory PVC was specifically diagnostic. Exceptionally, the disappearance of the reset phenomenon was also observed in the two cNVP-ORTs. Step 3 was verification of the AVN as the cNVP insertion site, evidenced by an atrial reset/block preceding the His reset/block in fast-slow AVNRT with a cNVP-SP and slow-fast AVNRT with a cNVP-FP or His reset preceding the atrial reset in slow-fast AVNRT with a cNVP-SP., Conclusion: AVNRT with a bystander cNVP can be diagnosed in the three steps with few exceptions. Notably, tachycardia reset/termination without atrial capture one cycle after delivery of a His-refractory PVC is specifically diagnostic., Competing Interests: The authors have no conflict of interest to declare., (© 2023 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of Japanese Heart Rhythm Society.)

Background: For lesion size prediction, each input parameter, including ablation energy (AE), and output parameter, such as impedance, is individually used. We hypothesize that using both parameters simultaneously may be more optimal., Methods and results: Radiofrequency applications at a range of power (30-50 W), contact force (10 g and 20 g), duration (10-60 s), and catheter orientation with normal saline (NS)- or half-normal saline (HNS)-irrigation were performed in excised porcine hearts. The correlations, with lesion size of AE, absolute impedance drop (∆Imp-drop), relative impedance drop (%Imp-drop), and AE*%Imp-drop were examined. Lesion size was analyzed in 283 of 288 lesions (NS-irrigation, n=142; HNS-irrigation, n=141) without steam pops. AE*%Imp-drop consistently showed the strongest correlations with lesion maximum depth (NS-irrigation, ρ=0.91; HNS-irrigation, ρ=0.94), surface area (NS-irrigation, ρ=0.87; HNS-irrigation, ρ=0.86), and volume (NS-irrigation, ρ=0.94; HNS-irrigation, ρ=0.94) compared with the other parameters. Moreover, compared with AE alone, AE*%Imp-drop significantly improved the strength of correlation with lesion maximum depth (AE vs. AE*%Imp-drop, ρ=0.83 vs. 0.91, P<0.01), surface area (ρ=0.73 vs. 0.87, P<0.01), and volume (ρ=0.84 vs. 0.94, P<0.01) with NS-irrigation. This tendency was also observed with HNS-irrigation. Parallel catheter orientation showed a better correlation with lesion depth and volume using ∆Imp-drop, %Imp-drop, and AE*%Imp-drop than perpendicular orientation., Conclusions: The combination of input and output parameters is more optimal than each single parameter for lesion prediction.

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a key role in the formation of flat symmetric leaves. AS2 represses the expression of the abaxial gene ETTIN / AUXIN RESPONSE FACTOR3 ( ETT / ARF3 ). AS2 interacts in vitro with the CGCCGC sequence in ETT / ARF3 exon 1. In cells of leaf primordia, AS2 localizes at peripheral regions of the nucleolus as two AS2 bodies, which are partially overlapped with chromocenters that contain condensed 45S ribosomal DNA repeats. AS2 contains the AS2/LOB domain, which consists of three sequences conserved in the AS2/LOB family: the zinc finger (ZF) motif, the ICG sequence including the conserved glycine residue, and the LZL motif. AS2 and the genes NUCLEOLIN1 ( NUC1 ), RNA HELICASE10 ( RH10 ), and ROOT INITIATION DEFECTIVE2 ( RID2 ) that encode nucleolar proteins coordinately act as repressors against the expression of ETT / ARF3. Here, we examined the formation and patterning of AS2 bodies made from as2 mutants with amino acid substitutions in the ZF motif and the ICG sequence in cells of cotyledons and leaf primordia. Our results showed that the amino acid residues next to the cysteine residues in the ZF motif were essential for both the formation of AS2 bodies and the interaction with ETT / ARF3 DNA. The conserved glycine residue in the ICG sequence was required for the formation of AS2 bodies, but not for the DNA interaction. We also examined the effects of nuc1 , rh10 , and rid2 mutations, which alter the metabolism of rRNA intermediates and the morphology of the nucleolus, and showed that more than two AS2 bodies were observed in the nucleolus and at its periphery. These results suggested that the patterning of AS2 bodies is tightly linked to the morphology and functions of the nucleolus and the development of flat symmetric leaves in plants.

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in angiosperms. The model liverwort Marchantia polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha and the significance of PTI in bryophytes have not yet been described. We here show that M. polymorpha has four lysin motif (LysM)-domain-containing receptor homologs, two of which, LysM-receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, are responsible for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and highlights conserved elements and new aspects of pattern-triggered immunity in land plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Maintaining stable and transient quiescence in differentiated and stem cells, respectively, requires repression of the cell cycle. The plant RETINOBLASTOMA-RELATED (RBR) has been implicated in stem cell maintenance, presumably by forming repressor complexes with E2F transcription factors. Surprisingly we find that mutations in all three canonical E2Fs do not hinder the cell cycle, but similarly to RBR silencing, result in hyperplasia. Contrary to the growth arrest that occurs when exit from proliferation to differentiation is inhibited upon RBR silencing, the e2fabc mutant develops enlarged organs with supernumerary stem and differentiated cells as quiescence is compromised. While E2F, RBR and the M-phase regulatory MYB3Rs are part of the DREAM repressor complexes, and recruited to overlapping groups of targets, they regulate distinct sets of genes. Only the loss of E2Fs but not the MYB3Rs interferes with quiescence, which might be due to the ability of E2Fs to control both G1-S and some key G2-M targets. We conclude that collectively the three canonical E2Fs in complex with RBR have central roles in establishing cellular quiescence during organ development, leading to enhanced plant growth., (© 2023. Springer Nature Limited.)

We experienced a case of lead-related SVC syndrome, which was successfully treated using unique transvenous lead extraction technique and endovascular stenting. This case also suggests that intravascular ultrasound facilitates decision-making on whether the interventionist should perform TLE alone or add stenting in case of a lead-related venous obstruction., Competing Interests: None., (© 2023 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of Japanese Heart Rhythm Society.)

Somatic polyploidization often increases cell and organ size, thereby contributing to plant biomass production. However, as most woody plants do not undergo polyploidization, explaining the polyploidization effect on organ growth in trees remains difficult. Here we developed a new method to generate tetraploid lines in poplars through colchicine treatment of lateral buds. We found that tetraploidization induced cell enlargement in the stem, suggesting that polyploidization can increase cell size in woody plants that cannot induce polyploidization in normal development. Greenhouse growth analysis revealed that radial growth was enhanced in the basal stem of tetraploids, whereas longitudinal growth was retarded, producing the same amount of stem biomass as diploids. Woody biomass characteristics were also comparable in terms of wood substance density, saccharification efficiency, and cell wall profiling. Our results reveal tetraploidization as an effective strategy for improving woody biomass production when combined with technologies that promote longitudinal stem growth by enhancing metabolite production and/or transport., Competing Interests: Conflict of interestWe have no conflict of interest., (© 2022 Japanese Society for Plant Biotechnology.)

A 70-year-old man was referred for evaluation and treatment of wide QRS complex tachycardia with left bundle branch block morphology. Electrocardiography showed atrial bigeminy with an alternating bundle branch block (ABBB) aberration. Atrial burst stimulation reproducibly demonstrated ABBB. What is the mechanism?, Competing Interests: The authors declare that they have no conflict of interest in this article., (© 2022 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of the Japanese Heart Rhythm Society.)

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying β-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A 74-year-old man was admitted for a second replacement of a transvenous implantable cardioverter-defibrillator (ICD) generator at 15 years after implantation. Postoperatively, electrocardiographic monitoring showed atrial lead malfunction. Chest X-ray revealed that the tip of the active fixation atrial lead was dislocated. We present a case of atrial lead dislodgement after a very long-term period of an ICD implantation., Competing Interests: The authors declare no conflict of interest for this article., (© 2021 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of Japanese Heart Rhythm Society.)

PurposeWe aimed to evaluate the extent of collagenase clostridium histolyticum (CCH) diffusion in Dupuytren's contracture (DC) for tissues outside of the contracture cord using Magnetic Resonance Imaging (MRI) immediately after CCH injection. Methods: 10 male patients aged 57-79 with DC of the metacarpophalangeal (MCP) joints were examined. Extension deficits were 10-60°(mean, 34.3) and 0-60°(mean, 26.6) in the MCP and proximal interphalangeal (PIP) joints, respectively. CCH injection was performed according to the standard method. MRI was performed within 15 min of CCH injection. Results: In all 10 cases, the extended area of high-intensity signal change outside of the cord was observed on short-T1 inversion recovery images (STIRs). Continuity from the insertion site was observed in the area of signal change involving the flexor tendon and neurovascular bundle. The signal change area spanned distally and proximally beyond the injection level. The signal change area expanded along the tendon sheath but no signal changes were observed inside the flexor tendon, suggesting the tendon sheath serves as a protective barrier from the CCH solution. After 1 week of injection, the mean decrease in contracture was 32.5°(94.7%) for the MCP joint and 19.8°(74.4%) for the PIP joint. In nine out of 10 cases, the extension deficit was within five degrees of full extension in the affected finger. There was no neurovascular injury or tendon rupture at 3 months of observation. Conclusions: MRI indicated the possible leakage of the drug outside of the cord during the early phase after administration, suggesting that CCH could persistently affect healthy tissues until CCH inactivates its enzyme process.

Leaves of Arabidopsis develop from a shoot apical meristem grow along three (proximal-distal, adaxial-abaxial, and medial-lateral) axes and form a flat symmetric architecture. ASYMMETRIC LEAVES2 ( AS2 ), a key regulator for leaf adaxial-abaxial partitioning, encodes a plant-specific nuclear protein and directly represses the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 ( ETT/ARF3 ). How AS2 could act as a critical regulator, however, has yet to be demonstrated, although it might play an epigenetic role. Here, we summarize the current understandings of the genetic, molecular, and cellular functions of AS2. A characteristic genetic feature of AS2 is the presence of a number of (about 60) modifier genes, mutations of which enhance the leaf abnormalities of as2 . Although genes for proteins that are involved in diverse cellular processes are known as modifiers, it has recently become clear that many modifier proteins, such as NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10), are localized in the nucleolus. Some modifiers including ribosomal proteins are also members of the small subunit processome (SSUP). In addition, AS2 forms perinucleolar bodies partially colocalizing with chromocenters that include the condensed inactive 45S ribosomal RNA genes. AS2 participates in maintaining CpG methylation in specific exons of ETT/ARF3 . NUC1 and RH10 genes are also involved in maintaining the CpG methylation levels and repressing ETT/ARF3 transcript levels. AS2 and nucleolus-localizing modifiers might cooperatively repress ETT/ARF3 to develop symmetric flat leaves. These results raise the possibility of a nucleolus-related epigenetic repression system operating for developmental genes unique to plants and predict that AS2 could be a molecule with novel functions that cannot be explained by the conventional concept of transcription factors.

Background: Sinus of Valsalva aneurysm (SVA) is a rare cardiac abnormality occurring in 0.09% of the general population, but few reports have examined its recurrence. Unruptured SVAs are usually asymptomatic., Case Summary: A 50-year-old woman presented with chest pain and a history of surgery for a ruptured right coronary SVA 32 years prior. Echocardiography showed the recurrence of an unruptured SVA of the non-coronary sinus with moderate aortic regurgitation, severe mitral regurgitation, and severe tricuspid regurgitation. Cardiac computed tomography (CT) revealed compression of the right coronary artery (RCA) between the SVA and sternum. Adenosine triphosphate stress myocardial perfusion imaging (MPI) identified reversible ischaemia of the inferior wall. The patient underwent patch closure of the SVA, aortic valve replacement, mitral valvuloplasty, and tricuspid annuloplasty. Post-operative MPI showed no residual ischaemia, and CT confirmed both successful repair of the SVA and intact RCA., Discussion: This case provides two noteworthy findings. First, the SVA recurred after 32 years. Second, a non-coronary SVA causing myocardial ischaemia is extremely rare given the long anatomical distance between the non-coronary sinus and coronary arteries. In our patient, the non-coronary SVA grew large enough within the anterior mediastinum to cause RCA compression., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts., (© The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Few studies have investigated the association between dietary intake and blood concentrations of water-soluble vitamins in patients with ulcerative colitis (UC). In the present study, vitamin concentrations were measured in the blood and urinary excretion of 23 outpatients with UC and compared against a control group of 20 healthy participants. A weighed food record procedure was used to ensure controlled macronutrient and vitamin intakes of the UC cohort. Individuals in the control group were given a semi-purified diet for 8 days prior to assessment. Multiple linear regression analysis was used to identify important differences in vitamin concentrations, independent of sex, age and other confounding variables. The blood concentrations of vitamins B 2 , C, niacin and folate were markedly lower in the patients with UC than those in the control group, and the renal clearance of vitamins B 1 , B 6 , B 12 and folate was notably higher in the UC cohort. It was concluded that vitamins B 2 , C, niacin and folate were at significantly lower concentrations in patients with UC following adjustment for coexisting factors. The lower levels of niacin may be partially due to impaired reabsorption. Chronic inflammation, common in patients with UC, with may contribute to the lower levels of other vitamins by rendering amino acid and carbohydrate metabolism into a hypermetabolic state. As the role of vitamins in metabolic activity is constant and pervasive, nutritional management including the application of water-soluble vitamins appears important for patients suffering from UC.

Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is expressed in developing and adult skeletal muscle and negatively regulates skeletal muscle growth. Recently, myostatin has been found to be expressed in tendons and increases tendon fibroblast proliferation and the expression of tenocyte markers. C2C12 is a mouse myoblast cell line, which has the ability to transdifferentiate into osteoblast and adipocyte lineages. We hypothesized that myostatin is capable of inducing tenogenic differentiation of C2C12 cells. We found that the expression of scleraxis, a tendon progenitor cell marker, is much higher in C2C12 than in the multipotent mouse mesenchymal fibroblast cell line C3H10T1/2. In comparison with other growth factors, myostatin significantly up-regulated the expression of the tenogenic marker in C2C12 cells under serum-free culture conditions. Immunohistochemistry showed that myostatin inhibited myotube formation and promoted the formation of spindle-shaped cells expressing tenomodulin. We examined signaling pathways essential for tenogenic differentiation to clarify the mechanism of myostatin-induced differentiation of C2C12 into tenocytes. The expression of tenomodulin was significantly suppressed by treatment with the ALK inhibitor SB341542, in contrast to p38MAPK (SB203580) and MEK1 (PD98059) inhibitors. RNAi silencing of Smad3 significantly suppressed myostatin-induced tenomodulin expression. These results indicate that myostatin has a potential role in the induction of tenogenic differentiation of C2C12 cells, which have tendon progenitor cell characteristics, through activation of Smad3-mediated signaling.

Agrobacterium-mediated transformation is a core technology for basic plant science and agricultural biotechnology. Improving transformation frequency is a major goal for plant transgenesis. We previously showed that T-DNA insertions in some histone genes decreased transformation susceptibility, whereas overexpression of several Arabidopsis H2A and H4 isoforms increased transformation. Overexpression of several histone H2B and H3 isoforms had little effect on transformation frequency. However, overexpression of histone H3-11 (HTR11) enhanced transformation. HTR11 is a unique H3 variant that lacks lysine at positions 9 and 27. The modification status of these lysine residues in canonical H3 proteins plays a critical role in epigenetic determination of gene expression. We mutated histone H3-4 (HTR4), a canonical H3.3 protein that does not increase transformation when overexpressed, by replacing either or both K9 and K27 with the amino acids in HTR11 (either K9I, K27Q, or both). Overexpression of HTR4 with the K27Q but not the K9I substitution enhanced transformation. HTR4 K27Q was incorporated into chromatin, and HTR4 K27Q overexpression lines exhibited deregulated expression of H3K27me3-enriched genes. These results demonstrate that mutation of K27 in H3.3 is sufficient to perturb H3K27me3-dependent expression in plants as in animals and suggest a distinct epigenetic role for histone HTR11. Further, these observations implicate manipulation of H3K27me3-dependent gene expression as a novel strategy to increase transformation susceptibility.

We examined the concentrations of water-soluble vitamins in blood and urinary excretion of 22 patients with type 2 diabetes mellitus (type 2DM) and 20 healthy control participants. Macronutrient and vitamin intakes of type 2DM subjects were measured using a weighed food record method. Control participants consumed a semipurified diet for eight days. Multiple linear regression models were used to determine whether significant differences existed in vitamin concentrations in blood independent of age, sex, and other confounding factors. Concentrations of vitamins B2, B6, C, niacin, and folate in blood were significantly lower in type 2DM subjects than in controls, independent of confounding factors. Renal clearances of vitamins B6, C, niacin, and folate were significantly higher in type 2DM subjects than in controls. In conclusion, concentrations of vitamins B2, B6, C, niacin, and folate in blood were significantly lower in type 2DM subjects than in controls, independent of confounding factors; based on the evidence of increased urinary clearance of these vitamins, the lower levels were likely due to impaired reabsorption processes., Competing Interests: Authors disclose no potential conflicts of interest.

This study aimed to compare the nutritional status and energy expenditure of hospitalized patients with Crohn's disease (CD) and those with ulcerative colitis (UC). Twenty-two hospitalized patients with CD and 18 patients with UC were enrolled in this study. We analyzed nutritional status upon admission by using nutritional screening tools including subjective global assessment, malnutrition universal screening tool, and laboratory tests. We measured resting energy expenditure (mREE) of the patients with indirect calorimetry and predicted resting energy expenditure (pREE) was calculated by using the Harris-Benedict equation. Results presented here indicate no significant difference in nutritional parameters and energy metabolism between CD and UC patients. In UC patients, a significant correlation was observed between mREE/body weight and disease activity detected by the Lichtiger and Seo indices. However, there was no correlation between mREE/body weight and Crohn's disease activity index in CD patients. Inflammatory cytokine interleukin-6 levels correlated with mREE/pREE in CD and UC patients while tumor necrosis factor-α was not. In conclusion, energy expenditure significantly correlated with disease activity in UC patients but not in CD patients. These results indicate that establishing daily energy requirements based on disease activity of UC is imperative for improving the nutritional status of patients.

We investigated the changes in energy expenditure during induction therapy in patients with severe or moderate ulcerative colitis. Thirteen patients (10 men, 3 women; mean age, 36.5 years) with ulcerative colitis admitted to the Shiga University Hospital were enrolled in this study. We measured the resting energy expenditure and respiratory quotients of these patients before and after induction therapy with indirect calorimetry. We analyzed the changes of nutritional status and serum inflammatory cytokine levels and also evaluated the relationship between energy metabolism and disease activity by using the Seo index and Lichtiger index. The resting energy expenditure was 26.3 ± 3.8 kcal/kg/day in the active stage and significantly decreased to 23.5 ± 2.4 kcal/kg/day after induction therapy (p<0.01). The resting energy expenditure changed in parallel with the disease activity index and C-reactive protein and inflammatory cytokine levels. The respiratory quotient significantly increased after induction therapy. Thus, moderate to severe ulcerative colitis patients had a hyper-metabolic status, and the energy metabolism of these patients significantly changed after induction therapy. Therefore, we recommend that nutritional management with 30-34 kcal/kg/day (calculated as measured resting energy expenditure × activity factor, 1.3) may be optimal for hospitalized ulcerative colitis patients.

Leaf primordia are generated at the periphery of the shoot apex, developing into flat symmetric organs with adaxial-abaxial polarity, in which the indeterminate state is repressed. Despite the crucial role of the ASYMMETRIC LEAVES1 (AS1)-AS2 nuclear-protein complex in leaf adaxial-abaxial polarity specification, information on mechanisms controlling their downstream genes has remained elusive. We systematically analyzed transcripts by microarray and chromatin immunoprecipitation assays and performed genetic rescue of as1 and as2 phenotypic abnormalities, which identified a new target gene, ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), which encodes an abaxial factor acting downstream of the AS1-AS2 complex. While the AS1-AS2 complex represses ETT by direct binding of AS1 to the ETT promoter, it also indirectly activates miR390- and RDR6-dependent post-transcriptional gene silencing to negatively regulate both ETT and ARF4 activities. Furthermore, AS1-AS2 maintains the status of DNA methylation in the ETT coding region. In agreement, filamentous leaves formed in as1 and as2 plants treated with a DNA methylation inhibitor were rescued by loss of ETT and ARF4 activities. We suggest that negative transcriptional, post-transcriptional and epigenetic regulation of the ARFs by AS1-AS2 is important for stabilizing early leaf partitioning into abaxial and adaxial domains.

It is necessary to use algorithms to analyze gene expression data from DNA microarrays, such as in clustering and machine learning. Previously, we developed the knowledge-based fuzzy adaptive resonance theory (KB-FuzzyART), a clustering algorithm suitable for analyzing gene expression data, to find clues for identifying gene networks. Leaf primordia form around the shoot apical meristem (SAM), which consists of indeterminate stem cells. Upon initiation of leaf development, adaxial-abaxial patterning is crucial for lateral expansion, via cellular proliferation, and the formation of flat symmetric leaves. Many regulatory genes that specify such patterning have been identified. Analysis by the KB-FuzzyART and subsequent molecular and genetic analyses previously showed that ASYMMETRIC LEAVES1 (AS1) and AS2 repress the expression of some abaxial-determinant genes, such as AUXIN RESPONSE FACTOR3 (ARF3)/ETTIN (ETT) and ARF4, which are responsible for defects in leaf adaxial-abaxial polarity in as1 and as2. In the present study, genetic analysis revealed that ARF3/ETT and ARF4 were regulated by modifier genes, BOBBER1 (BOB1) and ELONGATA3 (ELO3), together with AS1-AS2. We analyzed expression arrays with as2 elo3 and as2 bob1, and extracted genes downstream of ARF3/ETT by using KB-FuzzyART and molecular analyses. The results showed that expression of Kip-related protein (KRP) (for inhibitors of cyclin-dependent protein kinases) and Isopentenyltransferase (IPT) (for biosynthesis of cytokinin) genes were controlled by AS1-AS2 through ARF3/ETT and ARF4 functions, which suggests that the AS1-AS2-ETT pathway plays a critical role in controlling the cell division cycle and the biosynthesis of cytokinin around SAM to stabilize leaf development in Arabidopsis thaliana.

This study was to assess the resting energy expenditure of patients with esophageal cancer using indirect calorimetry. Eight male patients with esophageal cancer and eight male healthy controls were enrolled in this study. All patients underwent transthoracic esophagectomy with lymph nodes dissections. The resting energy expenditure was measured preoperatively, and on postoperative day 7 and 14 using indirect calorimetry. Preoperatively, the measured resting energy expenditure/body weight in these patients was significantly higher than that of the controls (23.3 ± 2.1 kcal/kg/day vs 20.4 ± 1.6 kcal/kg/day), whereas the measured/predicted energy expenditure from the Harris-Benedict equation ratio was 1.01 ± 0.09, which did not differ significantly from the control values. The measured resting energy expenditure/body weight was 27.3 ± 3.5 kcal/kg/day on postoperative day 7, and 23.7 ± 5.07 kcal/kg/day on postoperative day 14. Significant increases in the measured resting energy expenditure were observed on postoperative day 7, and the measured/predicted energy expenditure ratio was 1.17 ± 0.15. In conclusion, patients with operable esophageal cancers were almost normometabolic before surgery. On the other hand, the patients showed a hyper-metabolic status after esophagectomy. We recommended that nutritional management based on 33 kcal/body weight/day (calculated by the measured resting energy expenditure × active factor 1.2-1.3) may be optimal for patients undergoing esophagectomy.

We measured the energy expenditure weekly in patients undergoing a pylorus preserving pancreatoduodenectomy for bile duct cancer or pancreatic tumors. Twelve patients (5 women and 7 men; mean age 70.1 years) were enrolled in this study, and their resting energy expenditure levels were determined by indirect calorimetry. In these patients, a significant correlation was observed between the measured resting energy expenditures and the predicted resting energy expenditures calculated by the Harris-Benedict equation. The resting energy expenditures measured before surgery were almost the same as the predicted resting energy expenditures (measured resting energy expenditure: 22.4 ± 3.9 kcal/kg/day vs predicted resting energy expenditure: 21.7 ± 2.0 kcal/kg/day). The measured resting energy expenditure/predicted resting energy expenditure ratio, which reflects the stress factor, was 1.02 ± 0.10. After the pylorus preserving pancreatoduodenectomy, a significant increase in energy expenditure was observed, and the measured resting energy expenditure was 25.7 ± 3.5 kcal/kg/day on postoperative day 7 and 25.4 ± 4.9 kcal/kg/day on postoperative day 14. The measured resting energy expenditure/predicted resting energy expenditure ratio was 1.16 ± 0.14 on postoperative day 7, and 1.16 ± 0.18 on postoperative day 14 respectively. In conclusion, patients undergoing a pylorus preserving pancreatoduodenectomy showed a hyper-metabolic status as evaluated by their measured resting energy expenditure/predicted resting energy expenditure ratio. From our observations, we recommend that nutritional management based on 30 kcal/body weight/day (calculated by the measured resting energy expenditure×activity factor 1.2-1.3) may be optimal for patients undergoing a pylorus preserving pancreatoduodenectomy.

We investigated the energy expenditure in hospitalized patients with severe or moderate ulcerative colitis (UC), and compared them to healthy controls. Thirteen patients (5 women and 8 men; mean age 31.8 years; mean BMI 19.0 kg/m(2)) and 10 healthy volunteers were enrolled in this study. The resting energy expenditure (mREE) levels were determined by indirect calorimetry. The mREEs of the UC patients were significantly higher than those of healthy controls (26.4 +/- 3.6 vs 21.8 +/- 1.7 kcal/kg/day), although the mREEs of the UC patients were almost the same as the predicted REEs (pREEs) calculated by the Harris-Benedict equation (26.4 +/- 2.4 kcal/kg/day vs 26.5 +/- 2.6 kcal/kg/day). The mREE/pREE ratio, which reflects stress, was 1.0 +/- 0.15. In the UC patients, a significant correlation was observed between the mREEs and the clinical activity index. In conclusion, UC patients showed a hyper-metabolic status as evaluated by their mREE/body weight. Energy expenditure was significantly correlated with disease activity. From our observations, we recommend that nutritional management with more than 30-35 kcal/ideal body weight/day (calculated by the mREE x activity factor) may be optimal for active severe or moderate ulcerative colitis.

Aim: Adiponectin has insulin-sensitizing, anti-atherogenic, and anti-inflammatory properties, and researchers have recently reported that omega-3 polyunsaturated fatty acid (PUFA) can increase the serum adiponectin concentration, suggesting that dietary factors, such as fish intake, may have an influence on the serum adiponectin concentration. In general, Japanese subjects consume twice as much fish as people in other countries. We hypothesized that incremental change in serum omega-3 PUFA levels by fish intake is an important regulator of serum adiponectin even in Japanese subjects. The aim of this study was to explore the relationship among fish consumption, serum omega-3 PUFA, such as eicosapentaenoic acid (EPA), levels, and serum adiponectin levels., Method: We recruited 17 healthy Japanese volunteers (seven men and 10 women) for an 8-week fish-diet intervention (omega-3 PUFA 3.0 g/day) without affecting total energy intake, and measured serum adiponectin concentration and fatty acid profiles., Results: Fish-diet intervention significantly increased the serum adiponectin concentration in women (from 13.5+/-4.6 to 15.8+/-5.2 microg/mL, p <0.01) but not in men (from 8.7+/-2.8 to 8.7+/-2.5 microg/mL). Serum omega-3 PUFA increased more in female subjects than male subjects after the fish-diet intervention (57.3+/-86.6 vs 150.9+/-46.7 microg/mL, p=0.011), suggesting that changes in omega-3 PUFA concentration may explain the different response between sexes., Conclusion: A fish-based diet intervention increased the serum adiponectin concentration in young, non-obese, healthy Japanese female subjects. The increment in serum omega-3 PUFA may regulate the serum adiponectin concentration.

We investigated energy expenditure in hospitalized patients with Crohn's disease (CD), and determined optimal energy requirements for nutritional therapy. Sixteen patients (5 women and 11 men, mean age 36 year old, mean BMI 18.7 kg/m(2)) and 8 healthy volunteers were enrolled in this study. Measured resting energy expenditure (mREE) levels were determined by indirect calorimetry. The mREEs in CD patients were significantly higher than those of healthy controls (24.4 +/- 2.4 kcal/kg/day vs 21.3 +/- 1.7 kcal/kg/day). However, mREEs in CD patients were significantly lower than predicted REEs (pREEs) calculated by the Harris-Benedict equation (26.4 +/- 2.5 kcal/kg/day). Furthermore, mREE/pREE values were lower in undernourished patients than in well-nourished patients. CD patients had hyper-metabolic statuses evaluated by mREE/body weight, but increased energy expenditure did not contribute to weight loss in these patients. In conclusion, nutritional therapy with 25-30 kcal/ideal body weight/day (calculated by mREE x active factor) may be optimal for active CD patients, while higher energy intake values pose the risk of overfeeding.

The ASYMMETRIC LEAVES2 (AS2) gene is required for the generation of the flat and symmetrical shape of the leaf lamina in Arabidopsis. AS2 encodes a plant-specific protein with an AS2/LATERAL ORGAN BOUNDARIES (AS2/LOB) domain that includes a cysteine repeat, a conserved single glycine residue and a leucine-zipper-like sequence in its amino-terminal half. The Arabidopsis genome contains 42 genes, including AS2, that encode proteins with an AS2/LOB domain in their amino-terminal halves, and these genes constitute the AS2/LOB gene family. In the present study, we cloned and characterized cDNAs that covered the putative coding regions of all members of this family, and investigated patterns of transcription systematically in Arabidopsis plants. Comparisons among amino acid sequences that had been deduced from the cloned cDNAs revealed eight groups of genes, with two or three members each, and high degrees of identity among entire amino acid sequences, suggesting that some members of the AS2/LOB family might have redundant function(s). Moreover, no member of the family exhibited significant similarity, in terms of the deduced amino acid sequence of the carboxy-terminal half, to AS2. Results of domain swapping between AS2 and other members of the family showed that the AS2/LOB domain of AS2 cannot be functionally replaced by those of other members of the family, and that only a few dissimilarities among respective amino acid residues of the AS2/LOB domain of AS2 and those of other members are important for the specific functions of AS2.

Gene expression data obtained from DNA microarrays are very useful in revealing the mechanisms that drive life. It is necessary to analyze these data through the use of algorithms, as in clustering and machine-learning. In a previous study, we developed fuzzy adaptive resonance theory (FuzzyART) and applied it to gene expression data, to identify genetic networks. FuzzyART was used as a clustering algorithm that is very suitable for the analysis of biological data; however, although FuzzyART is very useful in the analysis of dozens of gene expression profiles, it is difficult to apply this method to thousands of gene expression profiles, owing to inherent category proliferation and long calculation time. In the present study, we developed a knowledge-based FuzzyART (KB-FuzzyART) to mitigate these problems. We first constructed a gene list-1 from the gene database of Arabidopsis thaliana as knowledge for KB-FuzzyART, because KB-FuzzyART requires any knowledge as input. This method was applied to gene expression data obtained via the microarray analysis of A. thaliana, to identify the downstream genes of ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2), both of which are involved in leaf development. The results of the analysis using KB-FuzzyART showed that the KNAT6 and YABBY5 (YAB5) genes are candidates for downstream factors, after a short calculation time for analysis. These results suggest that our gene list-1 is a very useful database for analyzing the expression profiles of genes that are related to the development of A. thaliana; they also suggest that the KB-FuzzyART has the high potential to function as a new method by which one can select candidate genes from thousands of genes, using gene expression data on mutant strains.

The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2. RNA1 encodes N-terminally overlapping replication proteins, p27 and p88, which are translated in a cap-independent manner. The 3' untranslated region of RNA1 contains RNA elements essential for cap-independent translation and negative-strand RNA synthesis. In this study, we investigated how p27 and p88 were engaged in the replication of RCNMV genomic RNAs by using DNA vectors or in vitro transcribed RNAs in protoplasts and in a cell-free extract of evacuolated BY-2 protoplasts. Our results show a cis-preferential requirement of p88, but not of p27, for the replication of RNA1. This mechanism might help to facilitate a switch in the role of RNA1 from mRNA to a replication template.

We investigated the trace element status in Crohn's disease (CD) patients receiving enteral nutrition, and evaluated the effects of trace element-rich supplementation. Thirty-one patients with CD were enrolled in this study. All patients were placed on an enteral nutrition regimen with Elental(R) (Ajinomoto pharmaceutical. Ltd., Tokyo, Japan). Serum selenium, zinc and copper concentrations were determined by atomic absorption spectroscopy. Serum selenoprotein P levels were determined by an ELISA system. Average serum levels of albumin, selenium, zinc and copper were 4.1 +/- 0.4 g/dl, 11.2 +/- 2.8 microg/dl, 71.0 +/- 14.8 microg/dl, and 112.0 +/- 25.6 microg/dl, respectively. In 9 patients of 31 CD patients, serum albumin levels were lower than the lower limit of the normal range. Serum selenium, zinc and copper levels were lower than lower limits in 12 patients, 9 patients and 1 patient, respectively. Serum selenium levels significantly correlated with both serum selenoprotein P levels and glutathione peroxidase activity. Supplementation of selenium (100 microg/day) and zinc (10 mg/day) for 2 months significantly improved the trace element status in CD patients. In conclusion, serum selenium and zinc levels are lower in many CD patients on long-term enteral nutrition. In these patients, supplementation of selenium and zinc was effective in improving the trace element status.

The ASYMMETRIC LEAVES2 (AS2) gene, a member of the AS2/LOB gene family, and the ASYMMETRIC LEAVES1 (AS1) gene of Arabidopsis thaliana participate in the development of a symmetrical, expanded lamina. We report here the patterns of expression of these genes, and the importance of the sites of such expression in leaf development. Transcripts of both genes accumulated in the entire leaf primordia at early stages, but the patterns of accumulation changed as the leaves expanded. AS2 and AS1 transcripts were detected, respectively, in the adaxial domain and in the inner domain between the adaxial and abaxial domains of leaves. The ratios of numbers of adaxial cells to abaxial cells in cotyledons of corresponding mutant lines were greater than the ratios in wild-type cotyledons. The low levels of ectopic expression of AS2 under the control of the AS1 promoter in as2 mutant plants restored an almost normal phenotype in some cases, but also resulted in flatter leaves than those of wild-type plants. Strong expression of the construct in wild-type and as2 plants, but not as1 plants, resulted in the formation of narrow, upwardly curled leaves. Our results indicate that AS2 represses cell proliferation in the adaxial domain in the presence of AS1, and that adaxial expression of AS2 at an appropriate level is critical for the development of a symmetrical, expanded lamina. Real-time RT-PCR analysis revealed that mutation of either AS2 or AS1 resulted in an increase in the levels of transcripts of ETTIN (ETT; also known as AUXIN RESPONSE FACTOR3, ARF3) and KANADI2 (KAN2), which are abaxial determinants, and YABBY5 (YAB5). Thus, AS2 and AS1 might negatively regulate the expression of these genes in the adaxial domain, which might be related to the development of flat and expanded leaves.

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.

The protein kinase cdc2 is conserved throughout eukaryotes and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. Here we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in lethality of the male gametophyte. Heterozygous plants produced mature siliques containing about 50% aborted seeds, and segregation distortion was observed in paternal inheritance. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The larger vegetative cell does not divide, but the smaller generative cell undergoes mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. The mutant sperm-like cell is fertile, and preferentially fuses with the egg cell to initiate embryogenesis. As the central cell nucleus remains unfertilized, however, double fertilization does not occur. In heterozygous plants, the embryo is arrested at the globular stage, most likely because of loss of endosperm development, whereas it is arrested at the one- or two-cell stage in presumptive homozygous plants. Thus, CDKA;1 is essential for cell division of the generative cell in male gametogenesis.

The ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana is involved in the establishment of the leaf venation system, which includes the prominent midvein, as well as in the development of a symmetric lamina. The gene product also represses the expression of class 1 knox homeobox genes in leaves. We have characterized the AS2 gene, which appears to encode a novel protein with cysteine repeats (designated the C-motif) and a leucine-zipper-like sequence in the amino-terminal half of the primary sequence. The Arabidopsis genome contains 42 putative genes that potentially encode proteins with conserved amino acid sequences that include the C-motif and the leucine-zipper-like sequence in the amino-terminal half. Thus, the AS2 protein belongs to a novel family of proteins that we have designated the AS2 family. Members of this family except AS2 also have been designated ASLs (AS2-like proteins). Transcripts of AS2 were detected mainly in adaxial domains of cotyledonary primordia. Green fluorescent protein-fused AS2 was concentrated in plant cell nuclei. Overexpression of AS2 cDNA in transgenic Arabidopsis plants resulted in upwardly curled leaves, which differed markedly from the downwardly curled leaves generated by loss-of-function mutation of AS2. Our results suggest that AS2 functions in the transcription of a certain gene(s) in plant nuclei and thereby controls the formation of a symmetric flat leaf lamina and the establishment of a prominent midvein and other patterns of venation.

The asymmetric leaves2 (as2) mutant of Arabidopsis thaliana generated leaf lobes and leaflet-like structures from the petioles of leaves in a bilaterally asymmetric manner. Both the delayed formation of the primary vein and the asymmetric formation of secondary veins were apparent in leaf primordia of as2 plants. A distinct midvein, which is the thickest vein and is located in the longitudinal center of the leaf lamina of wild-type plants, was often rudimentary even in mature as2 leaves. However, several parallel veins of very similar thickness were evident in such leaves. The complexity of venation patterns in all leaf-like organs of as2 plants was reduced. The malformed veins were visible before the development of asymmetry of the leaf lamina and were maintained in mature as2 leaves. In vitro culture on phytohormone-free medium of leaf sections from as2 mutants and from the asymmetric leaves1 (as1) mutant, which has a phenotype similar to that of as2, revealed an elevated potential in both cases for regeneration of shoots from leaf cells. Analysis by the reverse transcription-polymerase chain reaction showed that transcripts of the KNAT1, KNAT2 and KNAT6 (a recently identified member of the class 1 knox family) genes accumulated in the leaves of both as2 and as1 plants but not of wild type. Transcripts of the STM gene also accumulated in as1 leaves. These findings suggest that, in leaves, the AS2 and AS1 genes repress the expression of these homeobox genes, which are thought to maintain the indeterminate cell state in the shoot apical meristem. Taken together, our results suggest that AS2 and AS1 might be involved in establishment of a prominent midvein and of networks of other veins as well as in the formation of the symmetric leaf lamina, which might be related to repression of class 1 knox homeobox genes in leaves.