A gene-stacking approach to overcome the trade-off between drought stress tolerance and growth in Arabidopsis.

The molecular breeding of drought stress-tolerant crops is imperative for stable food and biomass production. However, a trade-off exists between plant growth and drought stress tolerance. Many drought stress-tolerant plants overexpressing stress-inducible genes, such as DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1A (DREB1A), show severe growth retardation. Here, we demonstrate that the growth of DREB1A-overexpressing Arabidopsis plants could be improved by co-expressing growth-enhancing genes whose expression is repressed under drought stress conditions. We used Arabidopsis GA REQUIRING 5 (GA5), which encodes a rate-limiting gibberellin biosynthetic enzyme, and PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which encodes a transcription factor regulating cell growth in response to light and temperature, for growth improvement. We observed an enhanced biomass and floral induction in the GA5 DREB1A and PIF4 DREB1A double overexpressors compared with those in the DREB1A overexpressors. Although the GA5 DREB1A double overexpressors continued to show high levels of drought stress tolerance, the PIF4 DREB1A double overexpressors showed lower levels of stress tolerance than the DREB1A overexpressors due to repressed expression of DREB1A. A multiomics analysis of the GA5 DREB1A double overexpressors showed that the co-expression of GA5 and DREB1A additively affected primary metabolism, gene expression and plant hormone profiles in the plants. These multidirectional analyses indicate that the inherent trade-off between growth and drought stress tolerance in plants can be overcome by appropriate gene-stacking approaches. Our study provides a basis for using genetic modification to improve the growth of drought stress-tolerant plants for the stable production of food and biomass.

Temporal and spatial changes in gene expression, metabolite accumulation and phytohormone content in rice seedlings grown under drought stress conditions.

In order to analyze the molecular mechanisms underlying the responses of plants to different levels of drought stress, we developed a soil matric potential (SMP)-based irrigation system that precisely controls soil moisture. Using this system, rice seedlings were grown under three different drought levels, denoted Md1, Md2 and Md3, with SMP values set to -9.8, -31.0 and -309.9 kPa, respectively. Although the Md1 treatment did not alter the visible phenotype, the Md2 treatment caused stomatal closure and shoot growth retardation (SGR). The Md3 treatment markedly induced SGR, without inhibition of photosynthesis. More severe drought (Sds) treatment, under which irrigation was terminated, resulted in the wilting of leaves and inhibition of photosynthesis. Metabolome analysis revealed the accumulation of primary sugars under Md3 and Sds and of most amino acids under Sds. The starch content was increased under Md3 and decreased under Sds. Transcriptome data showed that the expression profiles of associated genes supported the observed changes in photosynthesis and metabolites, suggesting that the time lag from SGR to inhibition of photosynthesis might lead to the accumulation of photosynthates under Md3, which can be used as osmolytes under Sds. To gain further insight into the observed SGR, transcriptome and hormonome analyses were performed in specific tissues. The results showed specific decreases in indole-3-acetic acid (IAA) and cytokinin levels in Md2-, Md3- and Sds-treated shoot bases, though the expression levels of hormone metabolism-related genes were not reflected in IAA and cytokinin contents. These observations suggest that drought stress affects the distribution or degradation of cytokinin and IAA molecules.

Double overexpression of DREB and PIF transcription factors improves drought stress tolerance and cell elongation in transgenic plants.

Although a variety of transgenic plants that are tolerant to drought stress have been generated, many of these plants show growth retardation. To improve drought tolerance and plant growth, we applied a gene-stacking approach using two transcription factor genes: DEHYDRATION-RESPONSIVE ELEMENT-BINDING 1A (DREB1A) and rice PHYTOCHROME-INTERACTING FACTOR-LIKE 1 (OsPIL1). The overexpression of DREB1A has been reported to improve drought stress tolerance in various crops, although it also causes a severe dwarf phenotype. OsPIL1 is a rice homologue of Arabidopsis PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), and it enhances cell elongation by activating cell wall-related gene expression. We found that the OsPIL1 protein was more stable than PIF4 under light conditions in Arabidopsis protoplasts. Transactivation analyses revealed that DREB1A and OsPIL1 did not negatively affect each other's transcriptional activities. The transgenic plants overexpressing both OsPIL1 and DREB1A showed the improved drought stress tolerance similar to that of DREB1A overexpressors. Furthermore, double overexpressors showed the enhanced hypocotyl elongation and floral induction compared with the DREB1A overexpressors. Metabolome analyses indicated that compatible solutes, such as sugars and amino acids, accumulated in the double overexpressors, which was similar to the observations of the DREB1A overexpressors. Transcriptome analyses showed an increased expression of abiotic stress-inducible DREB1A downstream genes and cell elongation-related OsPIL1 downstream genes in the double overexpressors, which suggests that these two transcription factors function independently in the transgenic plants despite the trade-offs required to balance plant growth and stress tolerance. Our study provides a basis for plant genetic engineering designed to overcome growth retardation in drought-tolerant transgenic plants.

The Arabidopsis transcriptional regulator DPB3-1 enhances heat stress tolerance without growth retardation in rice.

The enhancement of heat stress tolerance in crops is an important challenge for food security to facilitate adaptation to global warming. In Arabidopsis thaliana, the transcriptional regulator DNA polymerase II subunit B3-1 (DPB3-1)/nuclear factor Y subunit C10 (NF-YC10) has been reported as a positive regulator of Dehydration-responsive element binding protein 2A (DREB2A), and the overexpression of DPB3-1 enhances heat stress tolerance without growth retardation. Here, we show that DPB3-1 interacts with DREB2A homologues in rice and soya bean. Transactivation analyses with Arabidopsis and rice mesophyll protoplasts indicate that DPB3-1 and its rice homologue OsDPB3-2 function as positive regulators of DREB2A homologues. Overexpression of DPB3-1 did not affect plant growth or yield in rice under nonstress conditions. Moreover, DPB3-1-overexpressing rice showed enhanced heat stress tolerance. Microarray analysis revealed that many heat stress-inducible genes were up-regulated in DPB3-1-overexpressing rice under heat stress conditions. However, the overexpression of DPB3-1 using a constitutive promoter had almost no effect on the expression of these genes under nonstress conditions. This may be because DPB3-1 is a coactivator and thus lacks inherent transcriptional activity. We conclude that DPB3-1, a coactivator that functions specifically under abiotic stress conditions, could be utilized to increase heat stress tolerance in crops without negative effects on vegetative and reproductive growth.

Secondary Minimal Change Disease Due to Pancreatic Cancer Improved by Chemotherapy.

We herein describe an 82-year-old patient who presented with proteinuria and systemic edema. He was diagnosed with minimal change disease (MCD) and was found to have stage III pancreatic cancer. He could not undergo surgical resection due to invasion to the celiac artery and he was thus treated with chemotherapy. After a month of chemotherapy, his proteinuria improved to a normal level. After two months of chemotherapy, computed tomography indicated a partial response to the therapy. MCD can occur as paraneoplastic syndrome in patients with malignant disease, and chemotherapy can be effective for MCD associated with paraneoplastic syndrome.

Advanced Colon Cancer after Curative Resection of Intramucosal Adenocarcinoma with Endoscopic Submucosal Dissection.

Endoscopic resection, particularly endoscopic submucosal dissection (ESD), for colorectal cancers enables a precise pathological diagnosis and safe R0 resection. The recurrence rate after ESD is generally extremely low, with annual surveillance colonoscopy recommended. However, surveillance may not be considered for super-elderly patients owing to their condition. This is a case report of an 85-year-old man in whom curative resection was achieved for an intramucosal adenocarcinoma with ESD. The patient presented with a hypoechoic mass located in his lower right abdomen, diagnosed via surveillance abdominal ultrasound. He had undergone curative ESD for intramucosal cecal cancer 2 years prior. Colonoscopy revealed a type 2 epithelial tumor at the proximal aspect of the ESD scar. Ileocolic resection with lymph node dissection was performed. An epithelial tumor and well-differentiated adenocarcinoma but not a submucosal tumor was detected in the mucosal layer. The lesion was diagnosed not as a local recurrence after ESD but as a newly emerged original advanced cancer. After ESD for colorectal cancer, a newly developed advanced cancer may occur at the site of the ESD scar in a shorter term than usual. Surveillance colonoscopy after ESD is necessary even for super-elderly patients.

Analysis of Four Chitin-Active Lytic Polysaccharide Monooxygenases from Streptomyces griseus Reveals Functional Variation.

Lytic polysaccharide monooxygenases (LPMOs) are redox-active enzymes that cleave insoluble polysaccharides by an oxidative reaction. In the present study, we have characterized four recombinant putative chitin-active LPMOs from Streptomyces griseus (SgLPMO10B, -C, -D, and -F) and evaluated their potential in enhancing hydrolysis of α- and ß-chitin by three families of 18 chitinases of Serratia marcescens, SmChiA, -B, and -C. All four recombinant SgLPMO10s showed oxidative activity toward both α- and ß-chitin but exhibited different abilities to promote the release of chitobiose from chitin by chitinases depending on both the chitinase and the chitin type. These effects were observed under conditions where the amount of LPMO in the reaction was not rate-limiting, showing that the observed functional differences relate to different abilities of the LPMOs to interact with and act on the substrate. These results show that four seemingly similar LPMOs carrying out the same reaction, cleavage of chitin by C1 oxidation, may have different roles in natural chitin conversion, which provides a rationale for the multiplicity of these enzymes within the same organism. The ability of the LPMOs to act on more natural substrates was demonstrated by showing that SgLPMO10B improved chitin solubilization in dried powdered shrimp shells.

Hepatic methotrexate-associated lymphoproliferative disorders identified by multiple liver tumors: a case report and review of the literature.

BACKGROUND: Methotrexate, an immunosuppressant, is widely used as the standard therapeutic drug for rheumatoid arthritis. With the increasing frequency of use of methotrexate, adverse effects of methotrexate have been reported, one of which is known as methotrexate-associated lymphoproliferative disorders. The etiology of hepatic methotrexate-associated lymphoproliferative disorders remains largely unknown. To date, there have only been ten cases of hepatic methotrexate-associated lymphoproliferative disorders reported in the English literature and a case report is very rare. CASE PRESENTATION: An 82-year-old Japanese man with rheumatoid arthritis treated with methotrexate presented with fever. Contrast-enhanced computed tomography showed multiple hypovascular nodules in his liver, spleen, and lung, and para-aortic lesions. Endoscopic ultrasound-guided fine-needle aspiration biopsy for liver tumors was performed, and pathological results identified cluster of differentiation 20-positive lymphocytes. Discontinuance of methotrexate led to regression of the nodules and a final definitive diagnosis of methotrexate-associated lymphoproliferative disorders was made. CONCLUSIONS: We review 11 reported cases of hepatic methotrexate-associated lymphoproliferative disorders including the present case. Physicians should discontinue methotrexate in patients with rheumatoid arthritis treated with methotrexate when elevated soluble interleukin-2 receptor and hypovascular lesions in contrast-enhanced computed tomography are confirmed considering the possibility of methotrexate-associated lymphoproliferative disorders.

Fatal liver gas gangrene after biliary surgery.

INTRODUCTION: Liver gas gangrene is a rare condition with a highly mortality rate. It is mostly associated with host factors, such as malignancy and immunosuppression. PRESENTATION OF CASE: A 57-year-old female was admitted to our hospital with abnormalities of her serum hepato-biliary enzymes. She had a history of hypertension, diabetes mellitus, cerebral infarction, and chronic renal failure. She was diagnosed with bile duct cancer of the liver hilum and a left hepatectomy was carried out, with extrahepatic bile duct resection. Initially her post-operative state was uneventful. However, she suddenly developed melena with anemia on post-operative day (POD) 18. A Computed tomography (CT) examination on POD 19 revealed a massive build up of gas and portal gas formation in the anterior segment of the liver. Although we immediately provided the drainage and a probe laparotomy, she died on POD 20 due to shock with disseminated intravascular coagulation. DISCUSSION: Liver gas gangrene is rare and has a high mortality rate. This case seems to have arisen from an immunosuppressive state after major surgery with biliary reconstruction for bile duct cancer and subsequent gastrointestinal bleeding, leading to gas gangrene of the liver.

A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting α- and ß-chitin.

The lytic polysaccharide monooxygenases (LPMOs) have received considerable attention subsequent to their discovery because of their ability to boost the enzymatic conversion of recalcitrant polysaccharides. In the present study, we describe the enzymatic properties of SgLPMO10F, a small (15 kDa) auxilliary activity (AA) family 10 LPMO from Streptomyces griseus belonging to a clade of the phylogenetic tree without any characterized representative. The protein was expressed using a Brevibacillus-based expression system that had not been used previously for LPMO expression and that also ensures correct processing of the N-terminus crucial for LPMO activity. The enzyme was active towards both α- and ß-chitin and showed stronger binding and a greater release of soluble oxidized products for the latter allomorph. In chitinase synergy assays, however, SgLPMO10F worked slightly better for α-chitin, increasing chitin solubilization yields by up to 30-fold and 20-fold for α- and ß-chitin, respectively. Synergy experiments with various chitinases showed that the addition of SgLPMO10F leads to a substantial increase in the (GlcNAc)2 :GlcNAc product ratio, in reactions with α-chitin only. This underpins the structural differences between the substrates and also shows that, on α-chitin, SgLPMO10F affects the binding mode and/or degree of processivity of the chitinases tested. Variation in the only exposed aromatic residue in the substrate-binding surface of LPMO10s has previously been linked to preferential binding for α-chitin (exposed Trp) or ß-chitin (exposed Tyr). Mutation of this residue, Tyr56, in SgLPMO10F to Trp had no detectable effect on substrate-binding preferences but, in synergy experiments, the mutant appeared to be more efficient on α-chitin.