Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy.

Gene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at target sites in plants. Here we report an all-in-one approach of GT in Arabidopsis by combining a transcriptional and a translational enhancer for the Cas expression. We find that facilitating the expression of Cas9 and Cas12a variant by using enhancers can improve DSB and subsequent knock-in efficiency in the Arabidopsis genome. These results indicate that simply increasing Cas protein expression at specific timings - egg cells and early embryos - can improve the establishment of heritable GTs. This simple approach allows for routine genome engineering in plants.

Rice SUMOs and unification of their names.

Posttranslational modifications (PTMs) to proteins are regulatory mechanisms that play a critical role in regulating growth and development. The SUMO system is a rapid and dynamic PTM system employed by eukaryotic cells. Plant SUMOs are involved in many physiological processes, such as stress responses, regulation of flowering time and defense reactions to pathogen attack. In Arabidopsis thaliana and rice (Oryza sativa), eight and seven SUMO genes, respectively, were predicted by sequence analysis. Phylogenetic tree analysis of these SUMOs shows that they are divided into two groups. One consists of SUMOs that contain no SUMO acceptor site and are involved in monoSUMOylation of their target proteins. Rice OsSUMO1 and OsSUMO2 are in this group, and are structurally similar to each other and to Arabidopsis AtSUMO1. The other group is composed of SUMOs in which an acceptor site (ΨKXE/D) occurs inside the SUMO molecule, suggesting their involvement in polySUMOylation. Several studies on the rice SUMOs have been performed independently and reported. Individual names of rice SUMOs are confusing, because a unified nomenclature has not been proposed. This review clarifies the attribution of seven rice SUMOs and unifies the individual SUMO names.

Histone deacetylation regulates de novo shoot regeneration.

During de novo plant organ regeneration, auxin induction mediates the formation of a pluripotent cell mass called callus, which regenerates shoots upon cytokinin induction. However, molecular mechanisms underlying transdifferentiation remain unknown. Here, we showed that the loss of HDA19, a histone deacetylase (HDAC) family gene, suppresses shoot regeneration. Treatment with an HDAC inhibitor revealed that the activity of this gene is essential for shoot regeneration. Further, we identified target genes whose expression was regulated through HDA19-mediated histone deacetylation during shoot induction and found that ENHANCER OF SHOOT REGENERATION 1 and CUP-SHAPED COTYLEDON 2 play important roles in shoot apical meristem formation. Histones at the loci of these genes were hyperacetylated and markedly upregulated in hda19. Transient ESR1 or CUC2 overexpression impaired shoot regeneration, as observed in hda19. Therefore, HDA19 mediates direct histone deacetylation of CUC2 and ESR1 loci to prevent their overexpression at the early stages of shoot regeneration.

Peculiar properties of tuber starch in a potato mutant lacking the α-glucan water dikinase 1 gene GWD1 created by targeted mutagenesis using the CRISPR/dMac3-Cas9 system.

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze-thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

Efficiency of potato genome editing: Targeted mutation on the genes involved in starch biosynthesis using the CRISPR/dMac3-Cas9 system.

Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.

Species differences in liver microsomal hydrolysis of acyl glucuronide in humans and rats.

Acyl glucuronides (AGs) are known as one of the causes of idiosyncratic drug toxicity (IDT). Although AGs can be enzymatically hydrolysed by ß-glucuronidase and esterase, much information on their characteristics and species differences is lacking. This study was aimed to clarify species differences in AG hydrolysis between human and rat liver microsomes (HLM and RLM).To evaluate the AG hydrolysis profile, and the contribution of ß-glucuronidase and esterase towards AG hydrolysis in HLM and RLM, nonsteroidal anti-inflammatory drugs (NSAIDs) were used. AGs were incubated with 0.1 M Tris-HCl buffer (pH 7.4) and 0.3 mg/mL HLM or RLM in the absence or presence of ß-glucuronidase inhibitor, D-saccharic acid 1,4-lactone (D-SL) and esterase inhibitor, phenylmethylsulfonyl fluoride (PMSF).AGs of mefenamic acid (MEF-AG) and etodolac (ETO-AG) showed significantly higher AG hydrolysis rates in RLM than in HLM. Esterases were found to serve as AG hydrolases dominantly in HLM, whereas both esterases and ß-glucuronidase equally contribute to AG hydrolysis in RLM. However, MEF-AG and ETO-AG were hydrolysed only by ß-glucuronidase.We demonstrated for the first time that the activity of AG hydrolases towards NSAID-AGs differs between humans and rats.

Rice OsHAK5 is a major potassium transporter that functions in potassium uptake with high specificity but contributes less to cesium uptake.

Cesium (Cs) in the environment is primarily absorbed by a potassium (K) transporter. OsHAK5 is a KT/HAK/KUP family K-transporter showing a high affinity for K. We created cultured rice cells whose OsHAK5 was knocked down by RNAi (named KD). In the medium containing 1.0 m m and less K, the growth of KD was significantly suppressed, suggesting that OsHAK5 greatly contributed to K absorption under limited K conditions. Although Cs suppressed the growth of KD and WT, stronger inhibition was observed on KD. Both KD and WT accumulated similar amounts of Cs when they were cultured in a medium containing Cs, whereas lower amounts of K were detected in KD. These results suggest that OsHAK5 was less involved in the absorption of Cs, although it was essential to K absorption under limited K conditions. In contrast, this means that another transporter may contribute to cesium uptake in rice.

Distinct roles of HMGB1 in the regulation of P­glycoprotein expression in the liver and kidney of mice with lipopolysaccharide­induced inflammation.

The role of high mobility group box 1 (HMGB1) in the regulation of efflux transporters in the liver and kidney remains unclear, although it has been reported that HMGB1 can increase P­glycoprotein (P­gp) expression in the brain. The present study aimed to clarify the involvement of HMGB1 in the regulation of P­gp expression in the liver and kidney of mice with lipopolysaccharide (LPS)­induced inflammation. Mice were treated with LPS or LPS + glycyrrhizin (GL); GL is as an HMGB1 inhibitor. Subsequently, the expression levels of transporters, such as P­gp, and HMGB1 receptors, such as toll­like receptor (TLR)4 and receptor for advanced glycation end­products (RAGE), were determined by quantitative PCR and LC­MS/MS­based targeted proteomics. For the in vitro study, HepG2 and KMRC­1 cells were used, as was a co­culture of KMRC­1 and differentiated THP­1 cells. The mRNA and protein expression levels of Mdr1a and Tlr4 in the kidneys of LPS + GL­treated mice were significantly decreased compared with those in LPS mice. The results indicated that HMGB1 had little effect on the expression of Mdr1a and Tlr4 in the liver, since there was little change in of Mdr1a and Mdr1b expression between the LPS and LPS + GL­treated mice. Notably, regarding MDR1 mRNA expression, KMRC­1 cells were more responsive to LPS than HepG2 cells, and KMRC­1 cells treated with LPS exhibited increased levels compared with control KMRC­1 cells. In differentiated THP­1 cells, LPS treatment decreased the mRNA expression levels of TLR4, whereas they were restored to control levels by HMGB1. In conclusion, HMGB1 in the plasma and TLR4 in macrophages may be involved in the regulation of P­gp expression in the kidneys of inflamed mice.

Procedure for the efficient acquisition of progeny seeds from crossed potato plants grafted onto tomato.

Potato, Solanum tuberosum L. is an important crop. However, it is difficult to breed potato cultivars by applying conventional crossing methods because potato has a tetraploid genome and is vegetatively propagated. Flower formation and tuber development occur simultaneously. Many potato cultivars hardly produce any fruits after crossing and fail to produce seeds. We report an improved procedure for obtaining progeny seeds by grafting potatoes onto tomatoes. The rate of fruit formation was more than 19% when the grafted potatoes were used for the crossing experiments, whereas crossing using the ungrafted plants showed a rate of 1.1%. This result suggests that our procedure results in the easy acquisition of null-segregant progenies by crossing mutant lines. It is also expected to improve conventional potato breeding.

Slc25a39 and Slc25a40 Expression in Mice with Bile Duct Ligation or Lipopolysaccharide Treatment.

SLC25A39/40, involved in mitochondrial GSH (mGSH) import from the cytoplasm, is essential for protection against oxidative stress and mitochondrial dysfunction. We examined the effects of cholestasis, through bile duct ligation (BDL) and lipopolysaccharide (LPS)-induced inflammation in mice, on Slc25a39/40 expression. Additionally, we used human clear cell renal carcinoma (KMRC-1) cells to elucidate the mechanism of regulation of SLC25A39/40 expression in the kidneys after LPS treatment. BDL resulted in a decrease in Slc25a39 mRNA in the liver and a decrease in Slc25a39/40 mRNA and protein in the kidneys. Consequently, there was a significant decrease in mGSH levels in the kidneys of BDL mice compared with those in sham mice. LPS treatment resulted in increased Slc25a40 expression in the kidneys. In KMRC-1 cells, the combination treatment of LPS-RS or FPS-ZM1 with LPS suppressed the LPS-induced increase in SLC25A40, suggesting that SLC25A40 expression could be regulated by the signaling pathway via toll-like receptor 4 and the receptor for advanced glycation end products, respectively. Our findings contribute to understanding the role of mGSH in the maintenance of the mitochondrial redox state. To the best of our knowledge, this is the first study that demonstrates the changes in Slc25a39/40 expression in mice with cholestasis-associated renal injury and LPS-induced inflammation.

Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DIC) frequently induce drug-induced liver injury (DILI). It is unclear whether macrophages such as M1 and M2 participate in NSAID-associated DILI; elucidating this relationship could lead to a better understanding of the detailed mechanism of DILI. We co-cultured human hepatoma HepG2 cells with M1 or M2 derived from human monocytic leukemia THP-1 cells to examine the roles of M1 and M2 in DIC-induced cytotoxicity. DIC was added to the direct or indirect co-cultures of HepG2 cells with M1 or M2 (HepG2/M1 or HepG2/M2, respectively) at cell ratios of (1:0, 1:0.1, 1:0.4, and 1:1). In both direct and indirect HepG2/M2 co-cultures (1:0.4), there was lower lactate dehydrogenase release compared with HepG2/M1 co-cultures. Other NSAIDs as well as DIC showed similar protective effects of DIC-induced cytotoxicity. There were only slight differences in mRNA levels of apoptosis- and endoplasmic reticulum stress-associated factors between M1 and M2 after DIC treatment, suggesting that other factors determined the protective effects of M2 on DIC-induced cytotoxicity. Levels of high mobility group box 1 (HMGB1) in the medium and the mRNA expression levels of HMGB1 receptors were different between M1 and M2 after DIC treatment. Increased HMGB1 concentrations and expression of toll-like receptor 2 mRNA in M1 were observed compared with M2 after DIC treatment. In conclusion, these results suggested that the HMGB1/TLR2 signaling axis can be suppressed in M2 but not M1, leading to the different roles of M1 and M2 in NSAID-induced cytotoxicity.

Stereoselective Covalent Adduct Formation of Acyl Glucuronide Metabolite of Nonsteroidal Anti-Inflammatory Drugs with UDP-Glucuronosyltransferase.

A reactive metabolite of nonsteroidal anti-inflammatory drugs (NSAIDs), acyl-ß-D-glucuronide (AG), covalently binds to endogenous proteins. The covalent adduct formation of NSAIDs-AG may lead to the dysfunction of target proteins. Therefore, it is important to clarify the detailed characterization of the formation of covalent protein adducts of NSAID-AG. UDP-glucuronosyltransferase (UGT) catalyzes the conversion of NSAIDs to NSAIDs-AG. The aim of this study was to perform a quantitative analysis of the covalent adduct formation of NSAIDs-AG with UGT. Diclofenac-AG and ketoprofen-AG formed covalent adducts with organelle proteins. Next, the number of covalent adducts formed between NSAIDs-AG and UGT isoforms (UGT1A1, UGT1A9, UGT2B4, and UGT2B9) was determined. The capacity of diclofenac-AG to form covalent adducts with UGT1A9 or UGT2B7 was approximately 10 times higher than that of mefenamic acid-AG. The amounts of covalent adducts of AG of propionic acid derivative NSAIDs with UGT2B were higher than those with UGT1A. Stereoselectivity was observed upon covalent binding to UGT. A significant negative correlation between the half-lives of NSAIDs-AG in phosphate buffers and the amount of covalent adduct with UGT2B7 was observed, suggesting the more labile NSAID-AG forms higher irreversible bindings to UGT. This report provides comprehensive information on the covalent adduct formation of NSAIDs-AGs with UGT.

Relationship between the risk of idiosyncratic drug toxicity and formation and degradation profiles of acyl-glucuronide metabolites of nonsteroidal anti-inflammatory drugs in rat liver microsomes.

Acyl glucuronides (AGs) are considered to cause idiosyncratic drug toxicity (IDT), and evaluating the chemical instability of AGs may be useful for predicting the IDT risk of novel drug candidates. However, AGs show variations in their chemical instability, degree of formation, and enzymatic hydrolysis. Therefore, we evaluated the degree of AG formation, enzymatic hydrolysis, and chemical instability in liver microsomes and their relationship with IDT risk. Nonsteroidal anti-inflammatory drugs (NSAIDs) were classified into three categories in terms of their IDT risk as parent drugs: safe (SA), warning (WA), and withdrawn (WDN). To evaluate the enzymatic and non-enzymatic degradation of AG, the parent drugs were incubated with rat liver microsomes in the absence or presence of AG hydrolase inhibitors. The degree of AG formation and disappearance was considered as the rate constant. For all NSAIDs investigated, the number of AGs formed notably increased following addition of AG hydrolase inhibitors. Particularly, AG was produced by WDN drugs at a lower level than that produced by WA and SA drugs in the absence of AG hydrolase inhibitors but was significantly increased after adding AG hydrolase inhibitors. The rate constants of AG formation and non-enzymatic AG disappearance did not significantly differ among the WDN, WA, and SA drugs, whereas the rate constant of enzymatic AG disappearance of WDN drugs tended to be higher than those of WA and SA drugs. In conclusion, we evaluated the enzymatic degradation and chemical instability of AG by simultaneously producing it in liver microsomes. This method enables evaluation of AG degradation without preparing AG. Moreover, we determined the relationship between enzymatic AG degradation in rat liver microsomes and IDT risk.

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.

Assessment of hepatic prostaglandin E2 level in carbamazepine induced liver injury.

Objective. Carbamazepine (CBZ), a widely used antiepileptic drug, is one major cause of the idiosyncratic liver injury along with immune reactions. Conversely, prostaglandin E2 (PGE2) demonstrates a hepatoprotective effect by regulating immune reactions and promoting liver repair in various types of liver injury. However, the amount of hepatic PGE2 during CBZ-induced liver injury remains elusive. In this study, we aimed to evaluate the hepatic PGE2 levels during CBZ-induced liver injury using a mouse model. Methods. Mice were orally administered with CBZ at a dose of 400 mg/kg for 4 days, and 800 mg/kg on the 5th day. Results. Plasma alanine transaminase (ALT) level increased in some of mice 24 h after the last CBZ administration. Although median value of hepatic PGE2 amount in the CBZ-treated mice showed same extent as vehicle-treated control mice, it exhibited significant elevated level in mice with severe liver injury presented by a plasma ALT level >1000 IU/L. According to these results, mice had a plasma ALT level >1000 IU/L were defined as responders and the others as non-responders in this study. Even though, the hepatic PGE2 levels increased in responders, the hepatic expression and enzyme activity related to PGE2 production were not upregulated when compared with vehicle-treated control mice. However, the hepatic 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression and activity decreased significantly in responders when compared with control mice. Conclusions. These results indicate that elevated hepatic PGE2 levels can be attributed to the downregulation of 15-PGDH expression under CBZ-induced liver injury.

Creation of a potato mutant lacking the starch branching enzyme gene StSBE3 that was generated by genome editing using the CRISPR/dMac3-Cas9 system.

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.

Overexpression of rice OsLEA5 relieves the deterioration in seed quality caused by high-temperature stress.

Late embryogenesis abundant protein (LEA) genes are widely conserved in seed plant species and form a multigene family. While some LEAs are known to respond to environmental stresses, the function of many LEAs is unknown. OsLEA5 (Lea14A) interacts with a regulator of the endosperm storage production, FLO2, suggesting that OsLEA5 may be involved in endosperm quality control. RNAi knockdown line of OsLEA5 showed decreased seed weight. Transformant lines overexpressing OsLEA5 exhibited improved quality and seed weight of mature seeds when they were developed under high-temperature conditions, while seed quality strongly declined in wild-type plants exposed to high-temperature stress. These findings indicate that OsLEA5 contributes to suppressing the deterioration of seed quality when developed under high-temperature conditions.

Increased penetration of diphenhydramine in brain via proton-coupled organic cation antiporter in rats with lipopolysaccharide-induced inflammation.

Uptake transporters in brain microvascular endothelial cells (BMECs) are involved in the penetration of basic (cationic) drugs such as diphenhydramine (DPHM) into the brain. Lipopolysaccharide (LPS)-induced inflammation alters the expression levels and activities of uptake transporters, which change the penetration of DPHM into the brain. A brain microdialysis study showed that the unbound brain-to-plasma partition coefficient (K p,uu,brain) for DPHM in LPS rats was approximately two times higher than that in control rats. The transcellular transport of DPHM to BMECs was increased when BMECs were cultured with serum from LPS rats. Compared with control rats or BMECs, the brain uptake of DPHM in LPS rats was increased and the intracellular accumulation of DPHM was increased under a high intracellular pH in BMECs from LPS rats, respectively. Treatment of BMECs with transporter inhibitors or inflammatory cytokines had little impact on the intracellular accumulation of DPHM in BMECs. This study suggests that LPS-induced inflammation promotes unidentified proton-coupled organic cation (H+/OC) antiporters that improve the penetration of DPHM into rat brain via the blood-brain barrier.

Increased brain penetration of diphenhydramine and memantine in rats with adjuvant-induced arthritis.

Brain penetration of cationic drugs is an important determinant of their efficacy and side effects. However, the effects of alterations in the activity of uptake transporters in the brain under inflammatory conditions on the brain penetration of cationic drugs are not fully understood. The aim of this study was to examine changes in brain penetration of cationic drugs, including diphenhydramine (DPHM), memantine (MMT), and cimetidine (CMD), and changes in the expression of uptake transporters such as organic cation transporter (Oct) in brain microvascular endothelial cells (BMECs) under inflammatory conditions. To clarify the effects of inflammation on the brain penetration of DPHM, MMT, and CMD, we performed brain microdialysis studies in a rat model of adjuvant-induced arthritis (AA). Further, differences in transporter mRNA expression levels between BMECs from control and AA rats were evaluated. Brain microdialysis showed that the unbound brain-to-plasma partition coefficient (Kp,uu,brain) for DPHM and MMT was significantly lower in AA rats compared with control rats. OCT mRNA levels were increased and proton-coupled organic cation (H+/OC) antiporter mRNA levels were decreased in AA rats compared with control rats. Taken together, our findings suggest that inflammation decreases the brain penetration of H+/OC antiporter substrates such as DPHM and MMT.

Dissection of a rice OsMac1 mRNA 5' UTR to uncover regulatory elements that are responsible for its efficient translation.

The untranslated regions (UTRs) of mRNAs are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA has three splicing variants of the 5' UTR (UTRa, UTRb, and UTRc), which include a CU-rich region and three upstream open reading frames (uORFs). UTRc contains an additional 38-nt sequence, termed sp38, which acts as a strong translational enhancer of the downstream ORF; reporter analysis revealed translational efficiencies >15-fold higher with UTRc than with the other splice variants. Mutation analysis of UTRc demonstrated that an optimal sequence length of sp38, rather than its nucleotide sequence is essential for UTRc to promote efficient translation. In addition, the 5' 100 nucleotides of CU-rich region contribute to UTRc translational enhancement. Strikingly, three uORFs did not reveal their inhibitory potential within the full-length leader, whereas deletion of the 5' leader fragment preceding the leader region with uORFs nearly abolished translation. Computational prediction of UTRc structural motifs revealed stem-loop structures, termed SL1-SL4, and two regions, A and B, involved in putative intramolecular interactions. Our data suggest that SL4 binding to Region-A and base pairing between Region-B and the UTRc 3'end are critically required for translational enhancement. Since UTRc is not capable of internal initiation, we presume that the three-dimensional leader structures can allow translation of the leader downstream ORF, likely allowing the bypass of uORFs.