BackgroundPlant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.ResultsHere, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.ConclusionsWe show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.
BackgroundAge-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in the developed world. The two forms of advanced AMD, geographic atrophy and neovascular AMD, represent different pathological processes in the macula that lead to loss of central vision. Soft drusen, characterized by deposits in the macula without visual loss, are considered to be a precursor of advanced AMD. Recently, it has been proposed that a common missense variant, Y402H, in the Complement Factor H (CFH) gene increases the risk for advanced AMD. However, its impact on soft drusen, GA, or neovascular AMD—or the relationship between them—is unclear.Methods and FindingsWe genotyped 581 Icelandic patients with advanced AMD (278 neovascular AMD, 203 GA, and 100 with mixed neovascular AMD/GA), and 435 with early AMD (of whom 220 had soft drusen). A second cohort of 431 US patients from Utah, 322 with advanced AMD (244 neovascular AMD and 78 GA) and 109 early-AMD cases with soft drusen, were analyzed. We confirmed that the CFH Y402H variant shows significant association to advanced AMD, with odds ratio of 2.39 in Icelandic patients (p = 5.9 × 10−12) and odds ratio of 2.14 in US patients from Utah (p = 2.0 × 10−9) with advanced AMD. Furthermore, we show that the Y402H variant confers similar risk of soft drusen and both forms of advanced AMD (GA or neovascular AMD).ConclusionSoft drusen occur prior to progression to advanced AMD and represent a histological feature shared by neovascular AMD and GA. Our results suggest that CFH is a major risk factor of soft drusen, and additional genetic factors and/or environmental factors may be required for progression to advanced AMD.
The authors propose that a mitochondrial complex I defect is associated with the degeneration of TM cells in patients with POAG, and antioxidants and MPT inhibitors can reduce the progression of this condition.
A systematic study on the structure and function of Glucose-6-phosphate dehydrogenase (G6PD) variations was carried out in China. A total of 155,879 participants were screened for G6PD deficiency by the G6PD/6PGD ratio method and 6,683 cases have been found. The prevalence of G6PD deficiency ranged from 0 to 17.4%. With informed consent, 1,004 cases from 11 ethnic-based groups were subjected to molecular analysis. Our results showed the followings: (1) The G6PD variants are consistent across traditional ethnic boundaries, but vary in frequencies across ethnic-based groups in Chinese population, (2) The G6PD variants in Chinese population are different from those in African, European, and Indian populations, (3) A novel G6PD-deficiency mutation, 274C-->T, has been found, and (4) Denaturing high performance liquid chromatography is of great advantage to detecting G6PD-deficient mutations for diagnosis and genetic counseling. Moreover, functional analysis of the human G6PD variants showed the following: (1) The charge property, polarity, pK-radical and side-chain radical of the substituting amino acid have an effect on G6PD activity, (2) The G6PDArg459 and Arg463 play important roles in anchoring NADP+ to the catalytic domain to maintain the enzymatic activity, and (3) The sequence from codon 459 to the carboxyl terminal is essential for the enzymatic function.
Rationale:Peutz–Jeghers syndrome (PJS) is an autosomal dominant genetic syndrome characterized by a unique type of gastrointestinal hamartomatous polyp associated with oral and anal mucocutaneous pigmentations. Peutz–Jeghers polyps occur most numerously in the small intestine but frequently in the colon and stomach, only a few cases have been reported in the duodenum.Patient concern:A further family history survey discovered 10 out of 14 members of the family (in 4 generations) had mucocutaneous pigmentations, but many of them were living in rural areas where they had no access to specialized medical services, so none were checked with endoscopy for polyps of hamartoma.Diagnoses:We report the case of a boy patient with mucocutaneous pigmentations over the lips, and a history of recurrent bouts of vomit and anemia over the preceding two years, no abdominal pain and mass. An upper gastrointestinal endoscopy revealed some small polyps in the stomach and multiple sessile polyps in the second part of the duodenum, but colonoscopy exam did not reveal any lesion.Interventions:A double polypectomy and duodenum segmentary resection with end-to-end anastomosis was performed. Histopathology of the resected duodenum polyps indicated it to be a typical hamartomatous polyp.Outcomes:The child was under regular follow-up and recovered well.Lessons:In this case, the patient was characteristic with pigmentations on his lips and intermittent upper intestinal obstruction (due to mass duodenal polyps), there are no definitive guidelines for the treatment to duodenal PJS hamartomatous polyp, each case requires tailor-made management.
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