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.

     

Composition of uterine milk and its changes with gestational period in red stingrays (Hemitrygon akajei)

Uterine milk is secreted in the uterus for embryo nutrition in several elasmobranch species and may contribute to rapid embryonic growth, but the details of its composition and its functions are poorly understood. In this study, to explore the roles of uterine milk for embryos, its components throughout the gestational period were analysed in detail. Uterine milk was collected from pregnant red stingrays (Hemitrygon akajei) in the early, middle and late gestational periods, respectively (n= 3 for each period). The crude composition, constituent proteins and fatty acids in the milk were analysed. The uterine milk was rich in proteins throughout the gestational period, whereas lipids dramatically increased in the middle period and reduced slightly towards the late period. Some proteins potentially associated with nutrition, cartilage growth and embryonic immunity were found. Several enzymes related to central metabolism were also detected. The constituent fatty acids in the middle and late periods were similar to those in the egg yolks of elasmobranchs, except for C18:2, which was rich only in the uterine milk. The most abundant fatty acid in the milk was C16:1, which could function as a lipokine to promote lipid metabolism in the embryo. This study's data suggest that uterine milk may be secreted in addition to the egg yolk in elasmobranchs to support rapid and healthy embryonic growth.     

D1- and D2-like receptors differentially mediate the effects of dopaminergic transmission on cost–benefit evaluation and motivation in monkeys

It has been widely accepted that dopamine (DA) plays a major role in motivation, yet the specific contribution of DA signaling at D₁-like receptor (D₁R) and D₂-like receptor (D₂R) to cost–benefit trade-off remains unclear. Here, by combining pharmacological manipulation of DA receptors (DARs) and positron emission tomography (PET) imaging, we assessed the relationship between the degree of D₁R/D₂R blockade and changes in benefit- and cost-based motivation for goal-directed behavior of macaque monkeys. We found that the degree of blockade of either D₁R or D₂R was associated with a reduction of the positive impact of reward amount and increasing delay discounting. Workload discounting was selectively increased by D₂R antagonism. In addition, blocking both D₁R and D₂R had a synergistic effect on delay discounting but an antagonist effect on workload discounting. These results provide fundamental insight into the distinct mechanisms of DA action in the regulation of the benefit- and cost-based motivation, which have important implications for motivational alterations in both neurological and psychiatric disorders.

Using quantitatively controlled pharmacological manipulations, this study teases apart the role of D1- and D2-like dopamine receptors in motivation and goal-directed behavior in monkeys, revealing complementary roles of two dopamine receptor subtypes in the computation of the cost/benefit trade-off to guide action.     

Cleome hassleriana plants fully support the development and reproduction of Nesidiocoris tenuis

BioControl - Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is a zoophytophagous predator that feeds on plants as well as prey. Several non-crop host plant species have been used to maintain...     

Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H₂O₂, the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.     

Targeting Superoxide Dismutase to Renal Proximal Tubule Cells Attenuates Vancomycin-induced Nephrotoxicity in Rats

Vancomycin hydrochloride (VCM), a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). As it is known to induce renal dysfunction, the dose and the duration of its administration are limited. Moreover, the mechanism of VCM-induced renal dysfunction remains to be unclear. To evaluate the involvement of free radical on VCM-induced renal dysfunction, we carried out analysis with a hexamethylenediamine-conjugated superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Male Wistar rats (weighing 200-210 g) were intraperitonealy administered with 200 mg/kg of VCM twice a day for 7 days. AH-SOD 5 mg/kg/day was subcutaneously injected 5 min before every VCM injection. VCM induced renal injury dose-dependently. Biochemical analyses revealed that plasma levels of blood urea nitrogen and creatinine significantly increased in the VCM-treated group by an AH-SOD-inhibitable mechanism. VCM simultaneously elicited an increase of 8-OHdG levels and chemiluminescence intensity of free radical generation in the kidney. Histological examination revealed that VCM also elicited a marked destruction of glomeruli and necrosis of proximal tubules. AH-SOD inhibited these phenomena in the kidney. These results suggested that oxidative stress might underlie the pathogenesis of VCM-induced nephrotoxicity and targeting SOD and/or related antioxidants to renal proximal tubules might permit the administration of higher doses of VCM sufficient for eradication of MRSA without causing renal injury.     

Molecular Markers for Granulovacuolar Degeneration Are Present in Rimmed Vacuoles

Background

Rimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer''s disease and Parkinson''s disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers.

Methods

Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization.

Results

GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs.

Conclusions

These results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins.     

Genetic Structure of Wild Bonobo Populations: Diversity of Mitochondrial DNA and Geographical Distribution

Bonobos (Pan paniscus) inhabit regions south of the Congo River including all areas between its southerly tributaries. To investigate the genetic diversity and evolutionary relationship among bonobo populations, we sequenced mitochondrial DNA from 376 fecal samples collected in seven study populations located within the eastern and western limits of the species’ range. In 136 effective samples from different individuals (range: 7–37 per population), we distinguished 54 haplotypes in six clades (A1, A2, B1, B2, C, D), which included a newly identified clade (D). MtDNA haplotypes were regionally clustered; 83 percent of haplotypes were locality-specific. The distribution of haplotypes across populations and the genetic diversity within populations thus showed highly geographical patterns. Using population distance measures, seven populations were categorized in three clusters: the east, central, and west cohorts. Although further elucidation of historical changes in the geological setting is required, the geographical patterns of genetic diversity seem to be shaped by paleoenvironmental changes during the Pleistocene. The present day riverine barriers appeared to have a weak effect on gene flow among populations, except for the Lomami River, which separates the TL2 population from the others. The central cohort preserves a high genetic diversity, and two unique clades of haplotypes were found in the Wamba/Iyondji populations in the central cohort and in the TL2 population in the eastern cohort respectively. This knowledge may contribute to the planning of bonobo conservation.