Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

Inactivation of Rabbit Liver Carbonyl Reductase by Phenylglyoxal and 2,3,4-Trinitrobenzenesulfonate Sodium

The chemical modifications of rabbit liver carbonyl reductase (RLCR) with phenylglyoxal (PGO) and 2,3,4-trinitrobenzenesulfonate sodium (TNBS), which are respective chemical modifiers of arginine and lysine residues, were examined. RLCR was rapidly inactivated by these modifiers. Kinetic data for the inactivation demonstrated that each one of arginine and lysine residues is essential for catalytic activity of the enzyme. Furthermore, based on the protective effects of NADP +, NAD + and their constituents against the inactivation of RLCR by PGO and TNBS, we propose the possibility that the functional arginine and lysine residues are located in the coenzyme-binding domain of RLCR and interact with the 2′-phosphate group of NADPH.     

Site-Specific RNA Cleavage Using Terpyridine·Cu(II)-Linked 2′-O-Methyloligonucleotides

Abstract

2′-Deoxy- and 2′-O-methyl-5′-O-terpyridyl derivatives of adenosine and cytidine were synthesized and used to construct 5′-end-modified oligonucleotides. These antisense agents complexed with Cu(II) exclusively cleaved a complementary RNA oligomer at the site opposite the terpyridine-nucleoside residue. We also found that the terpyridine·Cu(II) moiety stabilizes 2′-O-methyl RNA duplex. These suggest that after RNA hybridization, the terpyridine moiety is close to the RNA strand, presumably in an end capping manner.     

Increase of Circulating CD11b+Gr1+ cells and Recruitment into the Synovium in Osteoarthritic Mice with Hyperlipidemia

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F₂ animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b⁺Gr1⁺ cells in spleens and peripheral blood was increased, and CD11b⁺Gr1⁺ cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b⁺Gr1⁺ cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b⁺Gr1⁺ cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F₂ mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b⁺Gr1⁺ cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b⁺Gr1⁺ cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.     

Plasma B-type Natriuretic Peptide as a Predictor of Cardiovascular Events in Subjects with Atrial Fibrillation: A Community-Based Study

Objectives

Atrial fibrillation (AF) is a significant public health issue due to its high prevalence in the general population, and is associated with an increased risk of cardiovascular (CV) events including systemic thrombo-embolism, heart failure, and coronary artery disease. The relationship between plasma B-type natriuretic peptide (BNP) and CV risk in real world AF subjects remains unknown.

Methods

The subject of the study (n = 228; mean age = 69 years) was unselected individuals with AF in a community-based population (n = 15,394; AF prevalence rate = 1.5%). The CV event free rate within each BNP tertile was estimated, and Cox regression analysis was performed to examine the relative risk of the onset of CV events among the tertiles. The prognostic ability of BNP was compared to an established risk score for embolic events (CHADS2 score). In addition, to determine the usefulness of BNP as a predictor in addition to CHADS2 score, we calculated Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) indices.

Results

During the follow-up period 58 subjects experienced CV events (52 per 1,000 person-years). The event-free ratio was significantly lower in the highest tertile (p < 0.02). After adjustment for established CV risk factors, the hazard ratio (HR) of the highest tertile was significantly higher than that of the lowest tertile (HR = 2.38; p < 0.02). The predictive abilities of plasma BNP in terms of sensitivity and specificity for general CV events were comparable to those of CHADS2 score. Adding BNP to the CHADS2 score only model improved the NRI (0.319; p < 0.05) and the IDI (0.046; p < 0.05).

Conclusion

Plasma BNP is a valuable biomarker both singly or in combination with an established scoring system for assessing general CV risk including stroke, heart failure and acute coronary syndrome in real-world AF subjects.     

Cyclic Phosphatidic Acid Stimulates cAMP Production and Inhibits Growth in Human Colon Cancer Cells

Colon cancer is a malignancy that develops in colon and rectal tissues. The prognosis for metastatic colon cancer remains poor, and novel therapeutic options are required to reduce colon cancer mortality. Recently, intracellular cAMP levels have been suggested to influence the behavior of cancer cells. Intriguingly, cyclic phosphatidic acid (cPA) and its structural analogs inhibit growth in many cancer cell lines, and our previous work has suggested that cPA increases cAMP production. Phosphodiesterase (PDE) type 3 isoforms PDE3A and PDE3B are expressed mainly in cardiovascular tissue and adipose tissue, respectively. Moreover, increase in intracellular cAMP levels has been associated with the inhibition of growth in colon cancer cells. These findings suggest that cPA could be used in colon cancer therapy. In this study, we found that cPA inhibited the growth of HT-29 cells, which express high levels of PDE3B, but not the growth of DLD-1 cells, which express low levels of PDE3B. Furthermore, cPA inhibited the phosphorylation of Akt in HT-29 cells in a dose-dependent fashion. Our results suggest that PDE3B expression and intracellular cAMP levels are correlated with the proliferation of colon cancer cells. These findings demonstrate for the first time that cPA may serve as a useful a molecule in targeted therapy for colon cancer.     

Crucial Residue Involved in L-Lactate Recognition by Human Monocarboxylate Transporter 4 (hMCT4)

Background

Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4.

Experimental approach

Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4.

Results

In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization.

Conclusions

Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.     

Colonization of vegetation-rich moraines and inference of multiple sources of colonization in the High Arctic for Salix arctica

Vegetation-rich patches in the High Arctic may serve as a significant source for vegetation reconstruction in the climate changes. Diversity and colonization, however, of such potential source populations in the High Arctic has rarely been studied. We examined chloroplast sequence variation in Salix arctica, a key species in the Canadian High Arctic, from four adjacent glacial moraines of differing ages on Ellesmere Island, Canada, as well as two other populations located at the center and southern end of the species’ range. The estimated ages of the moraines varied from 35,000 to 250 years old. The older moraine populations showed higher within-population genetic variation compared with the other moraine populations, which is generally attributed to differences in establishment age associated with plant densities among moraines. The moraines with smaller plant density had lower genetic diversity and had no private haplotypes, indicating the local population size and genetic diversity may not be recovered within a few thousand years. This suggests seed dispersal at a local scale may be limited even in species with high velocity of seed dispersal, and that High Arctic vegetation-rich patches may serve as significant source populations for sustaining local genetic diversity. In addition, the three regions we observed comprised an evolutionarily distinct lineage and significant population differentiation. This implies multiple sources for the colonization during the most recent deglaciation, resulting in the current wide distribution. Local as well as range-wide processes of colonization would be essential to understand vegetation responses in High Arctic to the environmental changes.