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Objective: To assess the impact of visual impairment (VI) on health-related quality of life and to compare the health burden of VI and other health conditions in Singapore., Design: Population-based cross-sectional study., Participants: We studied the 10 009 adults (3353 Chinese, 3397 Indians, and 3259 Malays) who underwent a comprehensive eye assessment and completed the European Quality of Life-5 Dimensions (EQ-5D) questionnaire in the Singapore Epidemiology of Eye Disease Study., Methods: We estimated the effects of VI, obesity, hypertension, diabetes, and hyperlipidemia on the EQ-5D index score using linear regression models and the association between VI and self-reported EQ-5D health problems using logistic regression models. We compared prevalence-based quality-adjusted life-year (QALY) loss associated with VI and other health conditions. For each condition, QALY loss was calculated for 100 000 persons in 1 year using associated reduction in EQ-5D index score estimated in regression analysis as disutility., Main Outcome Measures: The EQ-5D index score and annual QALY loss., Results: The EQ-5D index score decreased with increasing VI severity in all 3 ethnicities. For example, after adjusting for sociodemographic characteristics, the difference in EQ-5D index score between adults with bilateral severe VI and those without VI was -0.044 (95% confidence interval [CI], -0.089 to 0.001) in Chinese, -0.127 (95% CI, -0.237 to -0.017) in Indians, and -0.085 (95% CI, -0.148 to -0.022) in Malays. In all 3 ethnicities, VI was associated with reporting of problems in mobility (e.g., odds ratio [OR], 3.69 for Chinese with bilateral severe VI; 95% CI, 1.21-12.13) and usual activities (e.g., OR, 6.51 for Chinese with bilateral severe VI; 95% CI, 1.59-26.58). In Indians, VI was also associated with anxiety or depression (e.g., OR, 2.68 for bilateral severe VI; 95% CI, 1.11-6.50). The annual QALY loss associated with VI was 511.8 in Chinese, 608.8 in Indians, and 706.7 in Malays, greater than that associated with other health conditions examined in this study., Conclusions: Visual impairment is associated with substantial health burden among Asians in Singapore. The relatively high burden of VI highlights the importance of VI prevention. The ethnic difference exhibited in this burden warrants further study., (Copyright © 2014 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.

Wheat supplies about 20% of the total food calories consumed worldwide and is a national staple in many countries. Besides being a key source of plant proteins, it is also a major cause of many diet-induced health issues, especially celiac disease. The only effective treatment for this disease is a total gluten-free diet. The present report describes an effort to develop a natural dietary therapy for this disorder by transcriptional suppression of wheat DEMETER (DME) homeologs using RNA interference. DME encodes a 5-methylcytosine DNA glycosylase responsible for transcriptional derepression of gliadins and low-molecular-weight glutenins (LMWgs) by active demethylation of their promoters in the wheat endosperm. Previous research has demonstrated these proteins to be the major source of immunogenic epitopes. In this research, barley and wheat DME genes were cloned and localized on the syntenous chromosomes. Nucleotide diversity among DME homeologs was studied and used for their virtual transcript profiling. Functional conservation of DME enzyme was confirmed by comparing the motif and domain structure within and across the plant kingdom. Presence and absence of CpG islands in prolamin gene sequences was studied as a hallmark of hypo- and hypermethylation, respectively. Finally the epigenetic influence of DME silencing on accumulation of LMWgs and gliadins was studied using 20 transformants expressing hairpin RNA in their endosperm. These transformants showed up to 85.6% suppression in DME transcript abundance and up to 76.4% reduction in the amount of immunogenic prolamins, demonstrating the possibility of developing wheat varieties compatible for the celiac patients.

Myosin X (Myo10) with pleckstrin homology (PH) domains is a motor protein acting in filopodium initiation and extension. However, its potential role has not been fully understood, especially in neuronal development. In the present study the preferential accumulation of Myo10 in axon tips has been revealed in primary culture of hippocampal neurons with the aid of immunofluorescence from anti-Myo10 antibody in combination with anti-Tuj1 antibody as specific marker. Knocking down Myo10 gene transcription impaired outgrowth of axon with loss of Tau-1-positive phenotype. Interestingly, inhibition of actin polymerization by cytochalasin D rescued the defect of axon outgrowth. Furthermore, ectopic expression of Myo10 with enhanced green fluorescence protein (EGFP) labeled Myo10 mutants induced multiple axon-like neurites in a motor-independent way. Mechanism studies demonstrated that the recruitment of Myo10 through its PH domain to phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) was essential for axon formation. In addition, in vivo studies confirmed that Myo10 was required for neuronal morphological transition during radial neuronal migration in the developmental neocortex.

Background/aims: SM22α, transgelin, has been revealed to be specifically expressed in glomerular epithelial cells and interstitial cells, according to the nature of the renal injury. In this study, quantitative analyses of SM22α positivity were performed to investigate the pathological significance of its expression., Methods: Kidney samples of adriamycin nephropathy underwent immunohistochemistry with a newly established anti-SM22α monoclonal antibody. The SM22α positivity was quantified by an image analyzer. The correlation of the histological values with biochemical data was investigated statistically. Microstructural localization of SM22α was studied by immunoelectron microscopy., Results: SM22α was expressed along the dense basal microfilaments of degenerating podocytes, and diffusely in interstitial cells. Both the extent and intensity of SM22α expression in glomerular and tubulointerstitial area were correlated with the deterioration of renal function and the severity of proteinuria. Stepwise multiple linear regression analysis revealed that the extent of its positivity in glomerular or tubulointerstitial area was the determinant of the amount of proteinuria or the deterioration of creatinine clearance (Ccr), respectively. Inversely, the deterioration of Ccr was the most important predictor of SM22α expression., Conclusion: SM22α expression in podocytes and interstitial cells represented the severity of proteinuria and the deterioration of renal function. SM22α expression in renal tissues might be a hallmark of kidney diseases., (Copyright © 2011 S. Karger AG, Basel.)

Background: For efficient and large scale production of recombinant proteins in plants transient expression by agroinfection has a number of advantages over stable transformation. Simple manipulation, rapid analysis and high expression efficiency are possible. In pea, Pisum sativum, a Virus Induced Gene Silencing System using the pea early browning virus has been converted into an efficient agroinfection system by converting the two RNA genomes of the virus into binary expression vectors for Agrobacterium transformation., Results: By vacuum infiltration (0.08 Mpa, 1 min) of germinating pea seeds with 2-3 cm roots with Agrobacteria carrying the binary vectors, expression of the gene for Green Fluorescent Protein as marker and the gene for the human acidic fibroblast growth factor (aFGF) was obtained in 80% of the infiltrated developing seedlings. Maximal production of the recombinant proteins was achieved 12-15 days after infiltration., Conclusions: Compared to the leaf injection method vacuum infiltration of germinated seeds is highly efficient allowing large scale production of plants transiently expressing recombinant proteins. The production cycle of plants for harvesting the recombinant protein was shortened from 30 days for leaf injection to 15 days by applying vacuum infiltration. The synthesized aFGF was purified by heparin-affinity chromatography and its mitogenic activity on NIH 3T3 cells confirmed to be similar to a commercial product.

Aim: SM22α (transgelin) has been focused upon as a player in the process of phenotypic changes of types of cells. The SM22α expression in the rat anti-glomerular basement membrane (GBM) nephritis model and differences from an established phenotypic marker for the myofibroblast, α-smooth muscle actin (αSMA), were investigated., Methods: The rat kidney tissues were processed for histological studies, immunohistochemical and immunoelectronmicroscopy analyses on days 0, 7, 28, 42 and 56 after injection of rabbit anti-GBM serum for the disease induction., Results: Immunohistochemistry with anti-SM22α antibodies (Ab) revealed that kidneys of the nephritic rats on day 7 expressed SM22α in podocytes, crescentic cells and epithelial cells of Bowman's capsule. After 28 days, SM22α was also expressed in peritubular interstitial cells. Double immunofluorescence with anti-SM22α Ab and anti-αSMA Ab showed that SM22α was preferentially expressed in podocytes, whereas αSMA was positive in mesangial cells on day 7. After day 28, both molecules became positive in peritubular interstitial cells., Conclusion: SM22α was expressed in epithelial cells of inflamed glomeruli in the early phase, and then also in peritubular interstitial cells in the later phase of anti-GBM nephritis model. SM22α presented unique kinetics of expression distinct from αSMA., (© 2010 The Authors. Nephrology © 2010 Asian Pacific Society of Nephrology.)

Background/aims: SM22α, transgelin, is abundantly expressed in smooth muscle tissues and our previous work demonstrated that it is a novel marker of injured glomerular epithelial cells in rat antiglomerular basement membrane nephritis. In this study, we investigated SM22α expression in models of glomerular and interstitial renal injury., Methods: The 5/6 nephrectomy (Nx) model, ischemia-reperfusion (I/R) model and puromycin aminonucleoside (PAN) nephrosis of rats were studied. Immunohistochemical analyses and immunoelectron microscopic studies of SM22α expression were performed., Results: In the 5/6 Nx model, SM22α was first expressed in peritubular interstitial cells and was also expressed in injured glomerular epithelial cells at 8 weeks. In the I/R model, SM22α expression was induced in peritubular interstitial cells as early as 12 h after I/R with expression sustained at 7 days. However, SM22α was not detected in any glomerular cells or tubular epithelial cells. In PAN nephrosis, SM22α was only expressed in glomerular epithelial cells after 1 week, but expression was transient., Conclusion: SM22α was expressed in glomerular epithelial cells and interstitial cells in renal injury. SM22α is differentially upregulated in various models of renal injury and merits further study., (Copyright © 2010 S. Karger AG, Basel.)

More than 20 post-transcriptional gene silencing (PTGS) suppressors have been found since HC-Pro, the first gene silencing suppressor, was found in 1998. The silencing suppressor strongly suggested that gene silencing functions as natural defense mechanisms against viruses. It also represented a valuable tool for the dissection of the gene silencing pathway. We have used P1/HC-Pro RNA silencing suppressor activity to increase green fluorescent protein (GFP) expression in tobacco using an Agrobacterium-mediated transient expression system. P1/HC-Pro stimulated GFP-gene expression but not dsGFP-gene expression was shown by RT-PCR, Northern and Western blot analysis. Expression of the gene silencing suppressor and the target gene provided a new strategy of heterogeneous gene expressing in plants. It may be of commercial significance to produce foreign proteins using plant bioreactors.

Due to the laborious and scale-up limitation we have developed a simple system named "root absorption" to express foreign proteins in plants successfully. It has been shown that GFP was expressed in tobacco plants by root absorbing the Agrobacterium suspension containing TMV-based P35S-30B-GFP vector. Various factors influencing the gene expression were studied including Agrobacterium cell density, seedling age, plant materials and inoculation conditions. This system has the special advantages as simple and convenient work process, ease to scale-up and higher level of expression than leaf infiltration. Interestingly, GFP was expressed at 24h post-absorption. We assume that the root absorption system will facilitate the large-scale production of the recombinant pharmaceutical proteins in plants by means of transient expression.

The aFGF (acidic fibroblast growth factor) plays an important role in morphogenesis, angiogenesis and wound healing and is therefore of potential medical interest. A DNA fragment encoding haFGF (human aFGF) has been cloned into the PVX (potato virus X)-based binary vector (pgR107) and transiently expressed in Nicotiana benthamiana (a wild Australian tobacco) by agroinfection. Approx. 1 week after agroinfection, the recombinant haFGF accumulated in the agroinfected plants reached up to 1% of the total soluble protein. haFGF was then purified on heparin-Sepharose CL-6B. The purified haFGF could stimulate the growth of NIH 3T3 cells, suggesting that the recombinant haFGF expressed via PVX viral vector in N. benthamiana was active biologically.

Hairy roots induced by Agrobacterium rhizogenes grow faster, and are considered as genetically stable. These hairy roots can be used as an interesting material for the production of secondary metabolites of pharmaceutical value. Salidroside has been identified as the major compounds from the roots of Rhodiola sachalinensis A. BOR. Here, we provide an update that adds new perspectives on the prospects and challenges of producing Salidroside from hairy roots induced by Agrobacterium rhizogene in Rhodiola sachalinensis A. BOR. For high salidroside production, the optimal concentration for precursor (Tyrosol, Tyrosine, and Phenylalanine) and elicitor (Aspergillus niger, Coriolus versicolor, and Ganoderma lucidum) was added in the LB liquid medium, respectively. The addition of elicitor in the liquid MS medium and the utilization of precursor from chemical feeding enhanced biomass accumulation and salidroside production. The optimal concentration for elicitor and precursor in the liquid medium was 0.05 mg/l and 1 mmol/l, respectively.

Background/aims: Our previous comprehensive analysis of the genes expressed in kidneys with anti-glomerular basement membrane (GBM) nephritis using DNA microarrays showed that SM22alpha was one of the highly expressed genes. SM22alpha is a 22-kDa cytoskeletal protein that is exclusively expressed in smooth muscle cells. We investigated the localization of SM22alpha at mRNA and protein levels, and its pathological significance in anti-GBM nephritis kidneys., Methods: Northern blot analysis, in situ hybridization, immunohistochemistry and double immunofluorescence studies were performed. The specific antibody (Ab) against SM22alpha was obtained by immunization of rabbits with recombinant rat SM22alpha protein., Results: SM22alpha mRNA expression was upregulated in kidneys and inducibly expressed in the parietal and visceral glomerular epithelial cells in anti-GBM nephritis kidneys. Immunohistochemistry with anti-SM22alpha Ab showed that SM22alpha protein was localized in the same series of cells. Double immunofluorescence with anti-SM22alpha and anti-glomerular cell markers demonstrated that SM22alpha might be expressed in epithelial cells of injured glomeruli. In visceral epithelial cells, SM22alpha might be expressed in cells in which podocyte specific markers, podocalyxin and nephrin were lost., Conclusion: The injured glomerular epithelial cells in anti-GBM nephritis might undergo structural and functional alterations, including the expression of a smooth muscle marker, SM22alpha., (Copyright 2007 S. Karger AG, Basel.)

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth and typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT and 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants.

In the past 20 years, several systems have been developed to control transgene expression in plants using chemicals. The components used to construct these systems are derived from regulatory sequences mostly from non-plant organisms such as bacteria, fungi, insects and mammals. These constructs allowed transgene expression to be controlled temporally, spatially and quantitatively with the help of exogenous chemicals, without disturbing endogenous plant gene expression. Various chemically regulated transgene expression systems, their advantages/disadvantages and their potential for large-scale field application are reviewed.