Anthocyanin glucosylation mediated by a glycoside hydrolase family 3 protein in purple carrot.

Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP-sugar-dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl-glucose-dependent glucosyltransferase activity modifying cyanidin 3-xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3-type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells.

Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 µmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

Cervical ganglioneuroma arising from the dorsal root ganglion: illustrative case.

BACKGROUND: Ganglioneuroma is a benign and well-differentiated tumor derived from neural crest cells, which occurs infrequently, with most patients being female and adolescents. While predilection sites are the posterior mediastinum and retroperitoneal cavity, ganglioneuroma originating from the dorsal root ganglion is very rare. Here the authors report a case with C2 dorsal root ganglion-derived ganglioneuroma with some literature review. OBSERVATIONS: A 45-year-old male patient complained of persistent right-side throbbing occipital headache for more than a year. Magnetic resonance imaging (MRI) of the cervical spine revealed a dumbbell-shaped intradural extramedullary tumor from the C2 posterior surface of the odontoid to right C1-2 intervertebral foramen with high T2- and low T1-weighted signal intensities. The tumor displayed homogeneous contrast enhancement by MRI. The authors suspected schwannoma and performed a tumorectomy for both diagnosis and treatment purposes. Intraoperative findings showed that the tumor originated from the dorsal root ganglion, and pathological examination revealed ganglioneuroma. Immediately after the tumorectomy, the throbbing occipital headache disappeared and the patient was discharged from the hospital without major complications. LESSONS: Although ganglioneuroma derived from the dorsal root ganglion is very rare, a differential diagnosis of the ganglioneuroma should be made, when schwannoma is suspected.

Identification of Candidate Genes Responsible for Flower Colour Intensity in Gentiana triflora.

Gentians cultivated in Japan (Gentiana triflora and Gentiana scabra and hybrids) have blue flowers, but flower colour intensity differs among cultivars. The molecular mechanism underlying the variation in flower colour intensity is unclear. Here, we produced F2 progeny derived from an F1 cross of intense- and faint-blue lines and attempted to identify the genes responsible for flower colour intensity using RNA-sequencing analyses. Comparative analysis of flower colour intensity and transcriptome data revealed differentially expressed genes (DEGs), although known flavonoid biosynthesis-related genes showed similar expression patterns. From quantitative RT-PCR (qRT-PCR) analysis, we identified two and four genes with significantly different expression levels in the intense- and faint-blue flower lines, respectively. We conducted further analyses on one of the DEGs, termed GtMIF1, which encodes a putative mini zinc-finger protein homolog, which was most differently expressed in faint-blue individuals. Functional analysis of GtMIF1 was performed by producing stable tobacco transformants. GtMIF1-overexpressing tobacco plants showed reduced flower colour intensity compared with untransformed control plants. DNA-marker analysis also confirmed that the GtMIF1 allele of the faint-blue flower line correlated well with faint flower colour in F2 progeny. These results suggest that GtMIF1 is one of the key genes involved in determining the flower colour intensity of gentian.

[Lumbar Interbody Fusion:How to Achieve Reliable Bone Fusion?]

The goal of surgery for degenerative spine disease is to decompress nerves; however, extensive spinal decompression may compromise spinal stability. Therefore, spinal fusion surgery is performed to immediately stabilize such anatomical disruption during a short hospital stay and to allow quick recuperation. Recently, implants such as pedicle screws and intervertebral cages have been regularly used in lumbar fusion surgery. These implants are used to reconstruct the functional unit of the failed spine, correcting any deformity if necessary and maintaining its fixation until complete bone fusion. In other words, the essence of spinal fusion surgery is not the placement of implants but the induction of bone fusion. Therefore, each case requires a carefully developed surgical plan to achieve sufficient bone fusion for spinal stabilization. In this article, we describe the mechanism and the surgical technique for achieving reliable interbody fusion.

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian.

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

Synovial Cyst of the Atlantoaxial Joint Removed through a Posterior Intradural Approach.

Introduction. Synovial cysts rarely develop in the atlantoaxial joint. We report a case of posterior C1-2 laminectomy for a synovial cyst of the atlantoaxial joint which passed through the dorsal dura and put pressure on the cervical spinal cord. Case Presentation. A 62-year-old man with rapid progression of pain and weakness in the left upper extremity presented to our hospital. A cervical spine X-ray showed left C5-6 and C6-7 stenoses. A cervical magnetic resonance imaging showed an intradural extramedullary cystic lesion on the right side of the ventral cervical spinal cord at the C1-2 level and severe compression of the cervical spinal cord. Because a cyst was partially enhancing, a tumor lesion was not identifiable. Due to severe spinal cord compression, we performed intradural cyst removal via a posterior intradural approach with C1-2 laminectomy and left-sided C5-6 and C6-7 foraminotomies. One year after surgery, the cyst did not recur, and atlantoaxial instability did not appear. Discussion. A compressive lesion on the cervical spinal cord was not identified preoperatively as a synovial cyst. However, intraoperative and pathological findings suggested that the compressive lesion can be a synovial cyst which passed through the dorsal dura. The surgical treatment strategy for a synovial cyst of the atlantoaxial joint is controversial due to factors, such as the presence of atlantoaxial instability, level of cyst causing compression of the cervical spinal cord, severity of myelopathy, and cyst location. In the present study, the cervical spinal cord was highly compressed and the cyst was located on the right side of the cervical spinal cord; we chose cyst removal through a posterior intradural approach with C1-2 laminectomy.

Identification of the Biosynthetic Pathway for Anthocyanin Triglucoside, the Precursor of Polyacylated Anthocyanin, in Red Cabbage.

Red cabbage anthocyanin is utilized as a natural food colorant because of its stable and brilliant coloration. The major anthocyanin of red cabbage is cyanidin (Cy) mono- and di-acyltriglucoside; however, the biosynthetic pathway to generate this anthocyanin remains unclear. We isolated and identified four uridine diphosphate-glucose-dependent glucosyltransferase (UGT) cDNAs from red cabbage using RNA-seq. UGTs are involved in Cy triglucoside (CytriG) synthesis, the precursor of Cy acyltriglucoside. Enzymatic assays using recombinant proteins suggested that UGT78D5 encodes Cy 3GT, UGT79B45 encodes Cy 3-glucoside GT, UGT75C2 encodes Cy 3-sophoroside (Cy3Sp) 5GT, and UGT79B44 encodes flavonol 3-glucoside GT. Anthocyanin GT assays using crude proteins prepared from red cabbage suggested that CytriG is produced from intermediate products in the following order: Cy, Cy3G, Cy3Sp, and CytriG.

Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers.

Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3'-O-glycosyltransferase (Gt3'GT), and anthocyanin 5/3'-aromatic acyltransferase (Gt5/3'AT). The Gt5GT knockout lines accumulated delphinidin 3G, whereas the Gt3'GT knockout lines accumulated delphinidin 3G-5CafG as the major flower pigment. Knocking out Gt5/3'AT resulted in the accumulation of delphinidin 3G-5G-3'G and delphinidin 3G-5G as the primary and secondary pigments, respectively. These results indicated the existence of two pathways mediating the modification of delphinidin 3G-5G in flowers, with one involving a glycosylation by 3'GT and the other involving an acylation by 5/3'AT. The Gt5GT, Gt3'GT, and Gt5/3'AT transformants produced pale red violet, dull pink, and pale mauve flowers, respectively, unlike the vivid blue flowers of wild-type plants. Thus, the glycosylation and subsequent acylation of the 3'-hydroxy group of the B-ring in delphinidin aglycone is essential for the development of blue gentian flowers.

Carnation I locus contains two chalcone isomerase genes involved in orange flower coloration.

Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2'-glucoside (Ch2'G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2'G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2'G by chalcone 2'-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2'G simultaneously to give orange color.

Development of basic technologies for improvement of breeding and cultivation of Japanese gentian.

Japanese gentians are the most important ornamental flowers in Iwate Prefecture and their breeding and cultivation have been actively conducted for half a century. With its cool climate and large hilly and mountainous area, more than 60% of gentian production in Japan occurs in Iwate Prefecture. Recent advances in gentian breeding and cultivation have facilitated the efficient breeding of new cultivars; disease control and improved cultivation conditions have led to the stable production of Japanese gentians. Molecular biology techniques have been developed and applied in gentian breeding, including the diagnosis of viral diseases and analysis of physiological disorders to improve gentian production. This review summarizes such recent approaches that will assist in the development of new cultivars and support cultivation. More recently, new plant breeding techniques, including several new biotechnological methods such as genome editing and viral vectors, have also been developed in gentian. We, therefore, present examples of their application to gentians and discuss their advantages in future studies of gentians.

Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation.

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes.

Correction to: Effects of periodontal treatment on carotid intima-media thickness in patients with lifestyle-related diseases: Japanese prospective multicentre observational study.

Unfortunately, in Table-5 of the original article, the parameter in the 5th row was published incorrectly as "LDL-C (mg/dL)". The correct parameter should read as "HDL-C (mg/dL)".

Effects of periodontal treatment on carotid intima-media thickness in patients with lifestyle-related diseases: Japanese prospective multicentre observational study.

Atherosclerosis, a chronic inflammatory disease in arterial blood vessels, is one of the major causes of death in worldwide. Meanwhile, periodontal disease is a chronic inflammatory disease caused by infection with periodontal pathogens such as P. gingivalis (Porphyromonas gingivalis). Several studies have reported association between periodontal infection and atherosclerosis, but direct investigation about the effects of periodontal treatment on atherosclerosis has not been reported. We have planned Japanese local clinics to determine the relationship between periodontal disease and atherosclerosis under collaborative with medical and dental care. A prospective, multicentre, observational study was conducted including 38 medical patients with lifestyle-related diseases in the stable period under consultation at participating medical clinics and 92 periodontal patients not undergoing medical treatment but who were consulting at participating dental clinics. Systemic and periodontal examinations were performed before and after periodontal treatment. At baseline, LDL-C (low-density lipoprotein cholesterol) levels and percentage (%) of mobile teeth were positively related to plasma IgG (immunoglobulin) antibody titer against P. gingivalis with multivariate analysis. Corresponding to improvements in periodontal clinical parameters after treatment, right and left max IMT (maximum intima-media thickness) levels were decreased significantly after treatment (SPT-S: start of supportive periodontal therapy, SPT-1y: at 1 year under SPT, and SPT-3y: at 3 years under SPT). The present study has clarified our previous univariate analysis results, wherein P. gingivalis infection was positively associated with progression of atherosclerosis. Thus, routine screening using plasma IgG antibody titer against P. gingivalis and periodontal treatment under collaborative with medical and dental care may prevent cardiovascular accidents caused by atherosclerosis.

Biological effects of ion beam irradiation on perennial gentian and apple.

The development of new varieties of perennial plants generally requires lengthy and laborious procedures. In this study, we used ion beam irradiation mutagenesis in an attempt to accelerate the breeding process for perennial plants. We evaluated the biological effects of five ion beam sources (carbon, neon, argon, silicon, and iron) and neutron irradiation on Japanese gentian and apple. These treatments were applied at the National Institute of Radiological Sciences (NIRS) using the Heavy Ion Medical Accelerator in Chiba (HIMAC) and the Neutron-exposure Accelerator System for Biological Effect Experiments (NASBEE). Biological effects were observed in in vitro gentian plants after irradiation with ion beams at <10 Gy, whereas apple trees were less sensitive to ion beam irradiation. The growth of gentians in vitro was repressed by 3 Gy neutron irradiation, while that of grafted apple trees was not affected by 4 Gy neutron irradiation. During in vitro proliferation, seven pink-flowered lines were obtained from originally blue-flowered gentian after C and Ne ion beam irradiation treatments. Genomic and reverse transcription-PCR analyses of these lines suggested that the mutations occurred in the genomic region containing F3'5'H (encoding flavonoid 3',5'-hydroxylase). These results provide useful information for the mutagenesis and breeding of gentian, apple, and other perennial plants.

A Case of Atopic Myelitis with Cervical Cavernous Angioma.

Atopic myelitis, a type of myelitis which appears in patients with elevated serum levels of immunoglobulin E (IgE), occurs more commonly in the cervical spinal cord, but this mechanism has not yet been elucidated. Herein, we experienced a case of atopic myelitis developed during the growth of cervical cavernous angioma caused by bleeding. A 37-year-old woman suffered from hand swelling caused by a house cat licking. At the same time when cavernous angioma had grown, she experienced a numbness in her four extremities, and multifocal peritumoral hyperintense spinal cord signals were seen. The diagnosis of atopic myelitis was made because we observed significantly elevated levels of specific IgE antibody to cat dander. Symptoms disappeared immediately after steroid pulse therapy. We subsequently resected a cavernous angioma, and eosinophil invasion was found inside it. This is the first case report of atopic myelitis which developed in association with spinal cord vascular lesions. A local blood-brain barrier breakdown due to hemorrhagic lesions of the spinal cord may have contributed to the onset of atopic myelitis.

Surgical Treatment for Occipital Condyle Fracture, C1 Dislocation, and Cerebellar Contusion with Hemorrhage after Blunt Head Trauma.

Occipital condyle fractures (OCFs) have been treated as rare traumatic injuries, but the number of reported OCFs has gradually increased because of the popularization of computed tomography (CT) and magnetic resonance imaging (MRI). The patient in this report presented with OCFs and C1 dislocation, along with traumatic cerebellar hemorrhage, which led to craniovertebral junction instability. This case was also an extremely rare clinical condition in which the patient presented with traumatic lower cranial nerve palsy secondary to OCFs. When the patient was transferred to our hospital, the occipital bone remained defective extensively due to surgical treatment of cerebellar hemorrhage. For this reason, concurrent cranioplasty was performed with resin in order to fix the occipital bone plate strongly. The resin-made occipital bone was used to secure a titanium plate and screws enabled us to perform posterior fusion of the craniovertebral junction. Although the patient wore a halo vest for 3 months after surgery, lower cranial nerve symptoms, including not only neck pain but also paralysis of the throat and larynx, improved postoperatively. No complications were detected during outpatient follow-up, which continued for 5 years postoperatively.

TYK2 Promoter Variant and Diabetes Mellitus in the Japanese.

BACKGROUND: Recently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2). METHODS: Polymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese. FINDINGS: A TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody. INTERPRETATION: The TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans. FUNDING: Ministry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan.

[Juxta-facet Cyst Associated with Conjoined Nerve Roots:A Case Report].

We report a case of a patient with a juxta-facet cyst and conjoined nerve roots. A 66-year-old man presented with left leg pain from the past 4 months. Neurological examinations revealed L5 and S1 radiculopathy on the left side. Magnetic resonance imaging(MRI)detected a mass lesion located near the left intervertebral joint at the level of L5/S1 and canal stenosis at the level of L3/L4. A juxta-facet cyst was diagnosed by arthrography. We performed a curettage and resection of the mass, posterior lumbar interbody fusion at the level of L5/S1, and laminectomy at the level of L3/L4. Conjoined left L5/S1 nerve roots were observed during surgery. The patient recovered from the symptoms of L5 and S1 radiculopathy immediately after surgery. Postoperative review of the preoperative computed tomography images revealed bony abnormality in the L5/S1 joint. We speculate that the bony abnormality may be associated with the development of conjoined nerve roots and the juxta-facet cyst.

Novel C-xylosylflavones from the leaves and flowers of Iris gracilipes.

Two new C-glycosylflavones, apigenin 7,4'-dimethyl ether 6-C-ß-[(4"'-acetyl-L-rhamnopyranosyl)-(1-->2)-xylopyranoside] (1) and apigenin 7,4'-dimethyl ether 6-C-ß-L-rhamnopyranosyl-(1-->2)-xylopyranoside (2) were isolated from the leaves of Iris gracilipes (Iridaceae), along with two known flavonoids, swertiajaponin (3) and swertisin (4). C-Xylosylflavones 1 and 2 were elucidated by UV and NMR spectroscopy, mass spectrometry, and acid and alkaline hydrolyses. These novel compounds were also presented in the flowers.