Postharvest Properties of Ultra-Late Maturing Peach Cultivars and Their Attributions to Melting Flesh (M) Locus: Re-evaluation of M Locus in Association With Flesh Texture

The postharvest properties of two ultra-late maturing peach cultivars, "Tobihaku" (TH) and "Daijumitsuto" (DJ), were investigated. Fruit were harvested at commercial maturity and held at 25 degrees C. TH exhibited the characteristics of normal melting flesh (MF) peach, including rapid fruit softening associated with appropriate level of endogenous ethylene production In contrast, DJ did not soften at all during 3 weeks experimental period even though considerable ethylene production was observed. Fruit of TH and DJ were treated with 5,000 ppm of propylene, an ethylene analog, continuously for 7 days. TH softened rapidly whereas DJ maintained high flesh firmness in spite of an increase in endogenous ethylene production, suggesting that DJ but not TH lacked the ability to be softened in response to endogenous and exogenous ethylene/propylene. DNA-seq analysis showed that tandem endo-polygalacturonase (endoPG) genes located at melting flesh (M) locus, Pp-endoPGM (PGM), and Pp-endoPGF (PGF), were deleted in DJ. The endoPG genes at M locus are known to control flesh texture of peach fruit, and it was suggested that the non-softening property of DJ is due to the lack of endoPG genes. On the other hand, TH possessed an unidentified M haplotype that is involved in determination of MF phenotype. Structural identification of the unknown M haplotype, designated as M-0, through comparison with previously reported M haplotypes revealed distinct differences between PGM on M-0 haplotype (PGM-M-0) and PGM on other haplotypes (PGM-M-1). Peach M haplotypes were classified into four main haplotypes: M-0 with PGM-M-0; M-1 with both PGM-M-1 and PGF; M-2 with PGM-M-1; and M-3 lacking both PGM and PGF. Re-evaluation of M locus in association with MF/non-melting flesh (NMF) phenotypes in more than 400 accessions by using whole genome shotgun sequencing data on database and/or by PCR genotyping demonstrated that M-0 haplotype was the common haplotype in MF accessions, and M-0 and M-1 haplotypes were dominant over M-2 and M-3 haplotypes and co-dominantly determined the MF trait. It was also assumed on the basis of structural comparison of M haplotypes among Prunus species that the ancestral haplotype of M-0 diverged from those of the other haplotypes before the speciation of Prunus persica

Double non-contiguous fractures in a patient with spondylo-epiphyseal dysplasia with spinal ankylosis treated with open and percutaneous spinal fixation technique: a case report

Abstract Background Patients with ankylosing spines are susceptible to developing spinal fractures even with minor trauma and can develop early or late neurological injuries. These fractures require early and aggressive surgical management to enable spinal stability and/or neural decompression. Being highly unstable by nature, they require relatively long segment instrumentation and fusion, which can increase paravertebral soft tissue damage and perioperative bleeding. The purpose of this report is to describe a rare case of traumatic double fractures at the cervico-thoracic and thoraco-lumbar transition zones in ankylosing spine with spondylo-epiphyseal dysplasia (SED) of unknown cause, which were successfully treated with a combined open and percutaneous spinal fusion procedure. Case presentation A 46-year-old woman who was diagnosed with non-contiguous fractures in cervico-thoracic and thoraco-lumbar junction zones among multiple injuries sustained in a traffic accident was treated with hybrid techniques for posterior instrumentation with an open approach using a computed tomography (CT)-based navigation system and percutaneous pedicle-screwing method. She regained mobility to pre-admission levels and started walking on crutches 3 months postoperatively. Genetic testing for the cause of SED revealed no mutation in the COL2A1 or TRPVR4 genes. The union of fractured spine was confirmed on CT scan 1 year postoperatively. Conclusion This is the first report of double spinal fractures in an ankylosing spine with genetically undetermined spondyloepiphyseal dysplasia. A long-segment posterior instrumentation procedure incorporating the invasive treatment of spinal fractures in ankylosing spondylitis or diffuse idiopathic hyperostosis was effective

C/EBPβ directly bound to <i>Ihh</i> promoter.

<p>(A) EMSA for specific binding of C/EBPβ to the <i>Ihh</i> promoter. Consensus oligonucleotide (C), wild-type (WT) and mutant (MT) probes were incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Competition and supershift experiments were also performed. Data are representative of two independent experiments performed in duplicate. (B) A ChIP assay for C/EBPβ using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using primers as follows: promoter region of <i>Ihh</i> (from −259 to −160) and negative control (from −1274 and −1102 bp). Data are representative of two independent experiments performed in duplicate.</p

C/EBPβ binding element is crucial for RUNX2 to regulate transcriptional activity of <i>Ihh</i>.

<p>(A) Deletion constructs were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. ^*<i>p</i><0.05. (B) Mutation constructs of C/EBPβ and RUNX2 binding elements in pDel3 were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. ^*<i>p</i><0.05. (C) EMSA for specific binding of RUNX2 to the C/EBPβ binding site of <i>Ihh</i> promoter. Wild-type (WT) probe, which harbors C/EBPβ binding site, was incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Supershift experiment using RUNX2 antibody was also performed. Data are representative of two independent experiments performed in duplicate. (D) A ChIP assay for RUNX2 using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using same primers as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104547#pone-0104547-g005" target="_blank">Figure 5B</a>. Data are representative of two independent experiments performed in duplicate. (E) Immunoprecipitation (IP) and Immunoblotting were performed. Nuclear extract was obtained from C/EBPβ-transfected ATDC5 cells. Immunoprecipitated proteins with C/EBPβ, RUNX2 or IgG antibody were subjected to SDS-PAGE and immunoblotting using C/EBPβ or RUNX2 antibody.</p

C/EBPβ stimulated the expression of Ihh in <i>ex vivo</i> organ cultures.

<p><i>Ex vivo</i> organ culture of tibias dissected from E14.5 mouse embryos. Tibias were cultured for 4 days after transfection with adenovirus vectors expressing LacZ control (top row) and C/EBPβ-LAP (bottom row). Safranin O staining and immunofluorescent staining were performed to localize C/EBPβ, RUNX2 and Ihh. DAPI was used as a counterstain. Red, green and blue bars indicate the proliferative, pre-hypertrophic and hypertrophic zones, respectively. Scale bar, 500 µm. Histological analysis was repeated at least twice for each sample from six pairs of limbs, respectively.</p