MicroRNA159 Can Act as a Switch or Tuning MicroRNA Independently of Its Abundance in Arabidopsis

The efficacy of gene silencing by plant microRNAs (miRNAs) is generally assumed to be predominantly determined by their abundance. In Arabidopsis the highly abundant miRNA, miR159, acts as a molecular “switch” in vegetative tissues completely silencing the expression of two GAMYB-like genes, MYB33 and MYB65. Here, we show that miR159 has a diminished silencing efficacy in the seed. Using reporter gene constructs, we determined that MIR159 and MYB33 are co-transcribed in the aleurone and embryo of germinating seeds. However in contrast to vegetative tissues, MYB33 is not completely silenced. Instead, miR159 appears to shape the spatio-temporal expression pattern of MYB33 during seed germination. Transcript profiling in a time course during seed germination in wild-type and a mir159 mutant in which miR159 is almost absent, revealed that transcript levels of the GAMYB-like genes were similar between these two genotypes during germination, but much higher in the mir159 mutant once germination had completed. This attenuation in the silencing of the GAMYB-like genes was not explained by a decrease in mature miR159 levels, which remained constant at all time points during seed germination. We propose that miR159 acts as a tuner of GAMYB-like levels in Arabidopsis germinating seeds and that the activity of this miRNA is attenuated in the seed compared to vegetative tissues. This implies that the efficacy of miRNA-mediated silencing is not solely determined by miRNA abundance and target transcript levels, but is being determined through additional mechanisms

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31–46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course

Intraocular lenses in age-related macular degeneration

Purpose: The aim of this work is to review the lenses, assessing their advantages and disadvantages. We describe a total of seven types of intraocular lenses (IOLs) recommended for age-related macular degeneration (AMD). Methods: We used the PubMed web platform to search for implantable devices in various stages of AMD. We searched for both prospective and retrospective studies and also case reports. Results: Clinical results in AMD patients have been described for a total of seven types of IOLs recommended for AMD: an implantable miniature telescope (IMT), IOL-VIP System, Lipshitz macular implant (LMI), sulcus-implanted Lipshitz macular implant, LMI-SI, Fresnel Prism Intraocular Lens, iolAMD and Scharioth Macula Lens. Conclusions: We conclude that to objectively ascertain the effectiveness and safety of these lenses, further independent clinical studies with longer follow-up data are necessary prior to the general use of these optical devices

Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)

BACKGROUND: We have previously developed a long RT-PCR method for selective amplification of full-length PKD1 transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the PKD1 gene. These have provided us with an opportunity to study PKD1 mutations especially in its reiterated region which is difficult to examine. In this report, we have further developed the method of multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) for analysis of PKD1 mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and de novo PKD1 mutations are identified and reported. METHODS: Full-length PKD1 cDNA isolated from the patients with ADPKD was fractionated into nine overlapping segments by nested-PCR. Each segment was digested with sets of combined restriction endonucleases before the SSCP analysis. The fragments with aberrant migration were mapped, isolated, and sequenced. The presence of mutation was confirmed by the long-range genomic DNA amplification in the PKD1 region, sequencing, direct mutation detection, and segregation analysis in the affected family. RESULTS: Five PKD1 mutations identified are two frameshift mutations caused by two di-nucleotide (c. 5225_5226delAG and c.9451_9452delAT) deletions, a nonsense (Q1828X, c.5693C>T) mutation, a splicing defect attributable to 31 nucleotide deletion (g.33184_33214del31), and an in-frame deletion (L3287del, c.10070_10072delCTC). All mutations occurred within the reiterated region of the gene involving exons 15, 26, 15, 19 and 29, respectively. Three mutations (one frameshift, splicing defect, and in-frame deletion) are novel and two (one frameshift and nonsense) known. In addition, two mutations (nonsense and splicing defect) are possibly de novo. CONCLUSION: The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening PKD1 mutations in the full-length cDNA. Five mutations identified were all in the reiterated region of this gene, three of which were novel. The presence of de novo PKD1 mutations indicates that this gene is prone to mutations

Analysis of the Maize dicer-like1 Mutant, fuzzy tassel, Implicates MicroRNAs in Anther Maturation and Dehiscence

Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt) mutant has a mutation in dicer-like1 (dcl1), which encodes a key enzyme required for microRNA (miRNA) biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.ECU Open Access Publishing Support Fun

MicroRNAs play critical roles during plant development and in response to abiotic stresses

MicroRNAs (miRNAs) have been identified as key molecules in regulatory networks. The fine-tuning role of miRNAs in addition to the regulatory role of transcription factors has shown that molecular events during development are tightly regulated. In addition, several miRNAs play crucial roles in the response to abiotic stress induced by drought, salinity, low temperatures, and metals such as aluminium. Interestingly, several miRNAs have overlapping roles with regard to development, stress responses, and nutrient homeostasis. Moreover, in response to the same abiotic stresses, different expression patterns for some conserved miRNA families among different plant species revealed different metabolic adjustments. The use of deep sequencing technologies for the characterisation of miRNA frequency and the identification of new miRNAs adds complexity to regulatory networks in plants. In this review, we consider the regulatory role of miRNAs in plant development and abiotic stresses, as well as the impact of deep sequencing technologies on the generation of miRNA data

The methionine196arginine polymorphism in exon 6 of the tumor necrosis factor receptor 2 gene (TNFRSF1B) is associated with the polycystic ovary syndrome and hyperandrogenism.

Inflammatory cytokines such as TNFalpha may play a role in the pathogenesis of common metabolic disorders, including hyperandrogenism and the polycystic ovary syndrome (PCOS). The TNF receptor 2 mediates most of the metabolic effects of TNFalpha. In the present study, we have evaluated serum soluble TNF receptor 2 levels, and several common polymorphisms in the TNF receptor 2 gene (TNFRSF1B), in women presenting with PCOS or hyperandrogenic disorders. Initial studies included 103 hyperandrogenic patients (42 presenting with PCOS) and 36 controls from Spain. The 196R alleles of the M196R (676 T-G) variant in exon 6 of TNFRSF1B, which is in linkage disequilibrium with a CA-repeat microsatellite polymorphism in intron 4 of TNFRSF1B, tended to be more frequent in hyperandrogenic patients than in controls (P = 0.056), reaching statistical significance when the analysis was restricted to include only PCOS patients (P < 0.03). Extended analysis including another 11 hyperandrogenic patients from Spain and 64 patients and 29 controls from Italy confirmed the association between 196R alleles of the M196R variant and hyperandrogenic disorders (P < 0.05), which was maintained when restricting the analysis to PCOS patients (P < 0.02). On the contrary, the W-untranslated region (exon 10) variants 1663 G-A, 1668 T-G, and 1690 T-C were not associated with hyperandrogenism. The soluble TNF receptor 2 levels were not different between patients and controls but were increased in obese subjects, compared with lean individuals, and were affected by the interaction between the 1663 G-A and 1668 T-G variants in the W-untranslated region of TNFRSF1B. The TNFRSF1B genotype did not influence any clinical or biochemical variable related to hyperandrogenism or insulin sensitivity and was not associated with obesity, both in hyperandrogenic patients and healthy controls considered separately. In conclusion, the M196R (676 T-G) variant in exon 6 of TNFRSF1B is associated with hyperandrogenism and PCOS, further suggesting a role for inflammatory cytokines in the pathogenesis of these disorders