Identification and Characterization of PSEUDO-RESPONSE REGULATOR (PRR) 1a and 1b Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage (Brassica rapa L.).

The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, Brassica rapa PSEUDO-RESPONSE REGULATOR (BrPRR) 1a, 1b, and 1ab (both 1a and 1b double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9-16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2-4 different mutation types at the target sites in the BrPRR1a and BrPRR1b genes. To identify the functions of BrPRR1a and 1b genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, BrCCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and BrLHY (LATE ELONGATED HYPOCOTYL) a and b were examined under light/dark cycles and continuous light conditions. The BrPRR1a and 1ab double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the BrPRR1b mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in B. rapa, for both breeding and improvement.

Transcriptome and QTL mapping analyses of major QTL genes controlling glucosinolate contents in vegetable- and oilseed-type Brassica rapa plants.

Glucosinolates (GSLs) are secondary metabolites providing defense against pathogens and herbivores in plants, and anti-carcinogenic activity against human cancer cells. Profiles of GSLs vary greatly among members of genus Brassica. In this study, we found that a reference line of Chinese cabbage (B. rapa ssp. pekinensis), 'Chiifu' contains significantly lower amounts of total GSLs than the oilseed-type B. rapa (B. rapa ssp. trilocularis) line 'LP08'. This study aimed to identify the key regulators of the high accumulation of GSLs in Brassica rapa plants using transcriptomic and linkage mapping approaches. Comparative transcriptome analysis showed that, in total, 8,276 and 9,878 genes were differentially expressed between 'Chiifu' and 'LP08' under light and dark conditions, respectively. Among 162 B. rapa GSL pathway genes, 79 were related to GSL metabolism under light conditions. We also performed QTL analysis using a single nucleotide polymorphism-based linkage map constructed using 151 F5 individuals derived from a cross between the 'Chiifu' and 'LP08' inbred lines. Two major QTL peaks were successfully identified on chromosome 3 using high-performance liquid chromatography to obtain GSL profiles from 97 F5 recombinant inbred lines. The MYB-domain transcription factor gene BrMYB28.1 (Bra012961) was found in the highest QTL peak region. The second highest peak was located near the 2-oxoacid-dependent dioxygenase gene BrGSL-OH.1 (Bra022920). This study identified major genes responsible for differing profiles of GSLs between 'Chiifu' and 'LP08'. Thus, our study provides molecular insights into differences in GSL profiles between vegetative- and oilseed-type B. rapa plants.

Analysis of risk factors for lipid intolerance of intravenous fat emulsion in very low birth weight infants.

In order to prevent fatty acid deficiency and to supply enough energy, intravenous fat emulsion is necessary for parenteral nutrition in preterm neonates. However, parenteral administration of intravenous fat emulsion can induce lipid intolerance. The purpose of this study was to analyze risk factors for lipid intolerance in very low birth weight infants. This retrospective study included 80 preterm neonates whose birth weight was less than 1,500 g. Subjects were divided into 2 categories: those with a serum triglyceride level of ≥ 200 mg/dl (n = 33, 41%) and those with a serum triglyceride level of < 200 mg/dl (n = 47, 59%). We conducted logistic regression analysis using variables which were significant in univariate analysis. All statistical analyses were processed in SPSS version 19.0. Four risk factors for lipid intolerance were obtained through analysis of the electronic medical record. Lipid intolerance occurred more frequently in neonates with sepsis; those with a birth weight less than 1,000 g; those who was administered intravenous fat emulsion more than 2.6 g/kg/day; and those whose gestational age was less than 28 weeks. It is suggested that serum triglyceride levels should be closely monitored to prevent lipid intolerance in preterm neonates with the aforementioned characteristics.

Clinical and Pathological Features of Korean Patients with DNM2-Related Centronuclear Myopathy.

BACKGROUND AND PURPOSE: Centronuclear myopathy (CNM) is characterized by the presence of central nuclei within a large number of muscle fibers. Mutations of the dynamin 2 gene (DNM2) are common causes of autosomal dominant or sporadic CNM. The aim of this study was to characterize the clinical and pathological features of CNM relative to the presence of DNM2 mutations. METHODS: Six patients with clinical and pathological features of CNM were recruited. Detailed clinical and pathological findings were analyzed according to the presence of DNM2 mutations. RESULTS: We detected DNM2 mutations in four of the six sporadic CNM patients, and identified the following distinct clinical and pathological features in those patients with DNM2 mutations: preferential involvement of the distal lower limbs, typical nuclear centralization, and radially distributed sarcoplasmic strands in muscle pathology. In contrast, those without DNM2 mutations exhibited rather diffuse muscular involvement, and nuclear internalization and myofibrillar disorganization were more pronounced features of their muscle pathology. CONCLUSIONS: These findings suggest the presence of specific features in Korean CNM patients. A detailed clinical and pathological examination of CNM patients would be helpful for molecular genetic analyses of this condition.

Limb-girdle phenotype is frequent in patients with myopathy associated with GNE mutations.

The gene GNE encodes a bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Its mutations are found in distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM). Those disorders are characterized clinically by predominant anterior tibial muscle weakness and atrophy, and pathologically by rimmed vacuoles on muscle biopsy. We analyzed 11 Korean patients with GNE mutations. The mutations showed ethnic similarity to those of Japanese patients, showing the highest frequency with V572L. Another mutation of C13S was also found recurring in our patient group. Interestingly, about half of the patients showed limb-girdle myopathy rather than distal myopathy. This was further represented by limb muscle CT scans revealing atrophic hamstring and relatively spared anterior tibial muscle. However, quadriceps muscles were consistently spared both in distal and limb-girdle phenotypes. In conclusion, this study demonstrates a phenotypic diversity associated with GNE mutations. Recognizing a wider clinical spectrum of GNE mutations will help benefit more patients with imminent new therapy.

Mutations of CAPN3 in Korean patients with limb-girdle muscular dystrophy.

The limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessively inherited disease caused by a mutation of the calpain 3 gene (CAPN3), and is considered one of the most prevalent subtypes of limb-girdle muscular dystrophy (LGMD). In this study, we aimed to identify CAPN3 mutations and to characterize the phenotype of Korean patients with LGMD2A. Among 35 patients with LGMD, four patients, who showed calpain 3 deficiency on western blot analysis, were analyzed in this study. Total RNA extracted from frozen muscle tissue was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using six primer pairs covering all coding sequences of CAPN3, and direct sequencing was performed. Clinical and pathological features of the patients were also reviewed. We found four different mutations in five alleles from three patients. Of the pathogenic mutations identified, two were novel (c.2125T>C and c.2355-2357delTTC), and the others had been reported elsewhere (c.440G>C, c.1076C>T). All patients showed a high CK level with predominant proximal leg weakness, and the onset was in their childhood except for one patient. Among two novel CAPN3 mutations, one was a missense mutation (c.2125T>C [p.709Ser>Pro]), and the other was a small in-frame deletion causing omission of a single amino acid (c.2355-2357delTTC [p.786delPhe]). The clinical features of our patients were generally compatible with the characteristics of LGMD2A patients described in the previous studies.

A comparison of autogenous/reactive obsessions and worry in a nonclinical population: a test of the continuum hypothesis.

Employing the autogenous-reactive model of obsessions (Behaviour Research and Therapy 41 (2003) 11-29), this study sought to test a hypothesized continuum where reactive obsessions fall in between autogenous obsessions and worry with respect to several thought characteristics concerning content appraisal, perceived form, and thought triggers. Nonclinical undergraduate students (n=435) were administered an online packet of questionnaires designed to examine the three different types of thoughts. Main data analyses included only those displaying moderate levels of obsessions or worries (n=252). According to the most distressing thought, three different groups were formed and compared: autogenous obsession (n=34), reactive obsession (n=76), and worry (n=142). Results revealed that (a) relative to worry, autogenous obsessions were perceived as more bizarre, more unacceptable, more unrealistic, and less likely to occur; (b) autogenous obsessions were more likely to take the form of impulses, urges, or images, whereas worry was more likely to take the form of doubts, apprehensions, or thoughts; and (c) worry was more characterized by awareness and identifiability of thought triggers, with reactive obsessions through these comparisons falling in between. Moreover, reactive obsessions, relative to autogenous obsessions, were more strongly associated with both severity of worry and use of worrying as a thought control strategy. Our data suggest that the reactive subtype represents more worry-like obsessions compared to the autogenous subtype.