Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Genome-wide association studies identify loci controlling specialized seed metabolites in Arabidopsis.

Plants synthesize specialized metabolites to facilitate environmental and ecological interactions. During evolution, plants diversified in their potential to synthesize these metabolites. Quantitative differences in metabolite levels of natural Arabidopsis (Arabidopsis thaliana) accessions can be employed to unravel the genetic basis for metabolic traits using genome-wide association studies (GWAS). Here, we performed metabolic GWAS on seeds of a panel of 315 A. thaliana natural accessions, including the reference genotypes C24 and Col-0, for polar and semi-polar seed metabolites using untargeted ultra-performance liquid chromatography-mass spectrometry. As a complementary approach, we performed quantitative trait locus (QTL) mapping of near-isogenic introgression lines between C24 and Col-0 for specific seed specialized metabolites. Besides common QTL between seeds and leaves, GWAS revealed seed-specific QTL for specialized metabolites, indicating differences in the genetic architecture of seeds and leaves. In seeds, aliphatic methylsulfinylalkyl and methylthioalkyl glucosinolates associated with the ALKENYL HYDROXYALKYL PRODUCING loci (GS-ALK and GS-OHP) on chromosome 4 containing alkenyl hydroxyalkyl producing 2 (AOP2) and 3 (AOP3) or with the GS-ELONG locus on chromosome 5 containing methylthioalkyl malate synthase (MAM1) and MAM3. We detected two unknown sulfur-containing compounds that were also mapped to these loci. In GWAS, some of the annotated flavonoids (kaempferol 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-rhamnoside) were mapped to transparent testa 7 (AT5G07990), encoding a cytochrome P450 75B1 monooxygenase. Three additional mass signals corresponding to quercetin-containing flavonols were mapped to UGT78D2 (AT5G17050). The association of the loci and associating metabolic features were functionally verified in knockdown mutant lines. By performing GWAS and QTL mapping, we were able to leverage variation of natural populations and parental lines to study seed specialized metabolism. The GWAS data set generated here is a high-quality resource that can be investigated in further studies.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

Seed-coat protective neolignans are produced by the dirigent protein AtDP1 and the laccase AtLAC5 in Arabidopsis.

Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4' coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8' coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds.

Rituximab with standard LMB chemotherapy in pediatric high-risk mature B-cell non-Hodgkin lymphoma: A report from the JPLSG B-NHL14 trial.

BACKGROUND: The benefit of adding rituximab to standard lymphomes malins B (LMB) chemotherapy for children with high-risk mature B-cell non-Hodgkin lymphoma (B-NHL) has previously been demonstrated in an international randomized phase III trial, to which the Japanese Pediatric Leukemia/Lymphoma Study Group could not participate. METHODS: To evaluate the efficacy and safety of rituximab in combination with LMB chemotherapy in Japanese patients, we conducted a single-arm multicenter trial. RESULTS: In this study, 45 patients were enrolled between April 2016 and September 2018. A total of 33 (73.3%), 5 (11.1%), and 6 (13.3%) patients had Burkitt lymphoma/leukemia, diffuse large B-cell lymphoma, and aggressive mature B-NHL, not otherwise specified, respectively. Ten (22.2%) and 21 (46.7%) patients had central nervous system disease and leukemic disease, respectively. The median follow-up period was 47.5 months. Three-year event-free survival and overall survival were 97.7% (95% confidence interval, 84.9-99.7) and 100%, respectively. The only event was relapse, which occurred in a patient with diffuse large B-cell lymphoma. Seven patients (15.6%) developed Grade 4 or higher non-hematologic adverse events. Febrile neutropenia was the most frequent Grade 3 or higher adverse event after the pre-phase treatment, with a frequency of 54.5%. CONCLUSION: The efficacy and safety of rituximab in combination with LMB chemotherapy in children with high-risk mature B-NHL was observed in Japan.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.

Cloning and sequence analysis of 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase from Sarocladium kiliense U4520.

A novel enzyme, 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), was previously discovered from Sarocladium kiliense U4520. In order to identify the factors underlying the unique substrate specificity of IMM-4IH, we endeavored to determine the amino acid sequence of the enzyme. By comparing the partial amino acid sequence of the enzyme to whole genome sequencing data of S. kiliense U4520, the IMM-4IH gene was estimated. The putative gene was expressed in Pichia pastoris, and its activity and properties were found to be consistent with those of the native enzyme. Comparing the amino acid sequence of IMM-4IH with those in the CAZy database led to classification in the glycoside hydrolase family 49 (GH49). Several amino acids important for catalysis (Asp406, Asp425, and Asp426) and substrate recognition at subsites + 1 and -3 were estimated by multiple sequence alignment analysis. These results provide important information for characterizing IMM-4IH and other GH49 enzymes.

Synthesis of isomaltooligosaccharides using 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase.

Isomaltooligosaccharides (IMOs), including isomaltose, are valuable oligosaccharides, and the development of methods to synthesize high-purity IMOs has long been underway. We recently discovered a novel enzyme, 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), that showed promise for improving the synthesis process. In this study, we establish methods for synthesizing isomaltose and IMOs consisting of a variety of degrees of polymerization from starch using IMM-4IH. With 5% substrate, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75, the yield of isomaltose was 63.0%; incorporating isoamylase and cyclomaltodextrin glucanotransferase increased the yield to 75.3%. On the other hand, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710, IMOs were synthesized. The inclusion of isoamylase and α-amylase led to the 136 mM IMOs, consisting of oligosaccharides from isomaltose to isomaltodecaose, from 10% starch. The development of these efficient methods will be an important contribution to the industrial production of IMOs.

Fruit setting rewires central metabolism via gibberellin cascades.

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Publisher Correction: A cheminformatics approach to characterize metabolomes in stable-isotope-labeled organisms.

In the originally published Supplementary Information for this paper, the files presented as Supplementary Tables 3, 4, and 7 were duplicates of Supplementary Tables 5, 6, and 9, respectively. All Supplementary Table files are now correct online.

A cheminformatics approach to characterize metabolomes in stable-isotope-labeled organisms.

We report a computational approach (implemented in MS-DIAL 3.0; http://prime.psc.riken.jp/) for metabolite structure characterization using fully 13C-labeled and non-labeled plants and LC-MS/MS. Our approach facilitates carbon number determination and metabolite classification for unknown molecules. Applying our method to 31 tissues from 12 plant species, we assigned 1,092 structures and 344 formulae to 3,604 carbon-determined metabolite ions, 69 of which were found to represent structures currently not listed in metabolome databases.

Cryopreservation of paediatric ovarian tissue with an updated version of the Edinburgh criteria for appropriate patient selection.

RESEARCH QUESTION: Are the revised patient selection criteria for fertility preservation of children and adolescents appropriate? DESIGN: A retrospective and prospective observational cohort study implemented at a university hospital approved for fertility preservation by an academic society. The characteristics of children and the process of fertility preservation consultation were investigated. Mortality, the longitudinal course of the endocrine profile and the menstrual cycle were confirmed in patients who underwent ovarian tissue cryopreservation (OTC) before the age of 18 years. RESULTS: Of the 74 children and adolescents referred for a fertility preservation consultation, 40 (54.1%) had haematological disease, which included patients with rare diseases. The mean age of patients was 11.1 ± 4.3 years (median 12 years, range 1-17 years). In accordance with the revised criteria, 31 (41.9%) patients had their ovarian tissue cryopreserved. Two out of 31 had complications after surgery (infection and drug allergy) and one patient with leukaemia (3.2%) had minimum residual disease on the extracted ovarian tissue. Of the 14 patients (>12 years) who completed treatment, 12 (85.7%) had primary ovarian insufficiency (POI) more than a year after treatment. Two out of 31 (6.5%) died because of recurrence of their underlying disease (median 28 months, range 0-60 months). Oocyte cryopreservation, as an additional and salvage fertility preservation treatment, was suggested to five patients with biochemical status POI (procedures pending). CONCLUSION: The primary disease and patients' ages varied in fertility preservation for children and adolescents. Our patient selection criteria might be appropriate over a short follow-up period.

Outcome of children with relapsed high-risk neuroblastoma in Japan and analysis of the role of allogeneic hematopoietic stem cell transplantation.

BACKGROUND: In Japan, allogeneic hematopoietic stem cell transplantation is widely performed for recurrent neuroblastomas. This retrospective study aimed to investigate the prognosis of recurrent neuroblastoma in Japan and explore the effectiveness of allogeneic hematopoietic stem cell transplantation. METHODS: Clinical characteristics and data on the treatment of patients with high-risk neuroblastoma who experienced first progression between 2003 and 2010 after attaining complete remission or partial remission were collected from hospitals participating in the Japanese Neuroblastoma Research Group. RESULTS: Data from 61 patients who fulfilled these criteria were collected. The median interval from disease onset to first progression was 19 months (range, 7-65 months), whereas the median observation time of the surviving patients was 18 months (range, 1-69 months). All patients were treated with chemotherapy, where 22 and 3 patients received allogeneic and autologous hematopoietic stem cell transplantation, respectively. Seven patients were alive in second complete remission, and 39 died, including two in complete remission. The 3-year progression-free survival and overall survival rates were 15.3% (SE: 6.1%) and 16.9% (SE: 6.5%), respectively. For patients with allogeneic hematopoietic stem cell transplantation, the 3-year progression-free survival and overall survival were 28.3% (standard error, 12.0%) and 24.3% (standard error, 11.5%), respectively, and for patients without allogeneic hematopoietic stem cell transplantation, the 3-year progression-free survival and overall survival were 6.0% (standard error 5.5%) and 12.0% (standard error 7.6%), respectively. The duration of initial remission (≥ 18 months) and implementation of allogeneic hematopoietic stem cell transplantation were independently predictive of progression-free survival (P = 0.002 and P = 0.017), whereas for overall survival, only allogeneic hematopoietic stem cell transplantation was predictive (P = 0.012). CONCLUSION: Although allogeneic hematopoietic stem cell transplantation contributed to some improvement in prognosis, it was insufficient.

Characteristics of genetic alterations of peripheral T-cell lymphoma in childhood including identification of novel fusion genes: the Japan Children's Cancer Group (JCCG).

Peripheral T-cell lymphoma (PTCL) is a group of heterogeneous non-Hodgkin lymphomas showing a mature T-cell or natural killer cell phenotype, but its molecular abnormalities in paediatric patients remain unclear. By employing next-generation sequencing and multiplex ligation-dependent probe amplification of tumour samples from 26 patients, we identified somatic alterations in paediatric PTCL including Epstein-Barr virus (EBV)-negative (EBV- ) and EBV-positive (EBV+ ) patients. As recurrent mutational targets for PTCL, we identified several previously unreported genes, including TNS1, ZFHX3, LRP2, NCOA2 and HOXA1, as well as genes previously reported in adult patients, e.g. TET2, CDKN2A, STAT3 and TP53. However, for other reported mutations, VAV1-related abnormalities were absent and mutations of NRAS, GATA3 and JAK3 showed a low frequency in our cohort. Concerning the association of EBV infection, two novel fusion genes: STAG2-AFF2 and ITPR2-FSTL4, and deletion and alteration of CDKN2A/2B, LMO1 and HOXA1 were identified in EBV- PTCL, but not in EBV+ PTCL. Conversely, alterations of PCDHGA4, ADAR, CUL9 and TP53 were identified only in EBV+ PTCL. Our observations suggest a clear difference in the molecular mechanism of onset between paediatric and adult PTCL and a difference in the characteristics of genetic alterations between EBV- and EBV+ paediatric PTCL.

Diffuse Large B-Cell Lymphoma of the Trachea in a Child With Symptoms of Bronchial Asthma.

Tracheal tumors are rare in children and manifest symptoms of airway obstruction. A 14-year-old boy with a 5-month history of dyspnea and wheezing was referred to our hospital. Although he had been initially diagnosed with bronchial asthma, computed tomography revealed tracheal tumors. Histologic examination showed only necrotic tissue. Thereafter, the systemic steroid treatment for bronchial asthma was tapered off. A second computed tomography scan revealed new lesions in the pancreas and lung. Biopsy of the pancreatic lesion revealed a diffuse large B-cell lymphoma. The patient was administered standard chemotherapy, following which he went into complete remission.

Isolated Central Nervous System Progression During Systemic Treatment With Brentuximab Vedotin Monotherapy in a Pediatric Patient With Recurrent ALK-negative Anaplastic Large Cell Lymphoma.

Central nervous system (CNS) involvement in anaplastic large cell lymphoma (ALCL) is uncommon. CNS prophylaxis is not regularly included in second-line treatments for patients who develop CNS-negative relapses. We report a pediatric case of recurrent ALK-negative ALCL who developed isolated CNS progression during the treatment with brentuximab vedotin monotherapy. The patient achieved CNS remission after receiving the CNS-directed treatments including craniospinal irradiation. There is no evidence regarding whether brentuximab vedotin can cross the blood-brain barrier. CNS prophylaxis should be considered in high-risk patients with relapsed ALCL who receive second-line treatments containing agents with limited CNS penetration.

Efficient production of isomaltose and isomaltooligosaccharides from starch using 1,4-α-glucan 6-α-glucosyltransferase and isopullulanase.

We attempted to develop an efficient method for producing isomaltose, a disaccharide consisting of an α-(1→6)-linkage, from starch by combining enzymes of known activity. We found that the combination of 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75 and isopullulanase from Aspergillus brasiliensis ATCC 9642 led to the efficient synthesis of isomaltose. Inclusion of isoamylase and cyclomaltodextrin glucanotransferase resulted in increased efficiency, with production yields exceeding 70%. Furthermore, we considered that isomaltooligosaccharides could be synthesized from starch by combining 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710 and isopullulanase. In reactions that additionally utilized isoamylase and α-amylase, the total concentration of product, which included a series of isomaltooligosaccharides from isomaltose to isomaltodecaose, was 131 m m, and the ratio of 6-linked glucopyranosyl bonds to all bonds was 91.7% at a substrate concentration of 10%. The development of these manufacturing methods will accelerate the industrial production of isomaltose and isomaltooligosaccharides.

Cloning of the cycloisomaltotetraose-forming enzymes using whole genome sequence analyses of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006.

We performed whole genome sequence analyses of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006 that secrete enzymes to produce cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran. Full-length amino acid sequences of CI4-forming enzymes were identified by matching known N-terminal amino acid sequences with products of the draft genome. Domain searches revealed that the CI4-forming enzymes are composed of Glycoside Hydrolase family 66 (GH66) domain, Carbohydrate Binding Module family 35 (CBM35) domain, and CBM13 domain, categorizing the CI4-forming enzymes in the GH66. Furthermore, the amino acid sequences of the two CI4-forming enzymes were 71% similar to each other and up to 51% similar to cycloisomaltooligosaccharide glucanotransferases (CITases) categorized in GH66. Differences in sequence between the CI4-forming enzymes and the CITases suggest mechanisms to produce specific cycloisomaltooligosaccharides, and whole genome sequence analyses identified a gene cluster whose gene products likely work in concert with the CI4-forming enzymes.

Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006.

Glucanotransferases that can synthesize cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.