Search

Nitrogen (N) deposition and precipitation reduction shift soil organic carbon (SOC) turnover and can change the intensity of SOC stabilization pathways in the aggregate system. This study aimed to reveal the effect of 10 y of N access (50 kg N ha−1 yr−1 (N50)) and precipitation reduction (−30%, (PREC)) simulated in mixed temperate forest, with the predominance of Korean pine (Pinus koraiensis), on SOC stabilization pathways using a13C natural abundance approach. Control, N addition, PREC, and their interaction (PREC + N50) were carried out in a randomized complete block design with split plots. PREC increased SOC compared to the control because of the accumulation of coarse particulate organic matter (CPOM) and microbial residues by 12–147%. In contrast, bulk SOC was unaffected by N50 and PREC + N50; only a decrease in the C content in microaggregates occluded in macroaggregate fractions (−20%), and an increase in microbial residues of bulk soil was found. Based on the 13C natural abundance, C pathways were from microaggregates to macroaggregates and from CPOM to the mineral-associated organic matter in macroaggregates. The intensity of the C flows decreased in the PREC, with an increase of plant-derived C within aggregates in the CPOM. The intensity of the C flows increased in N50 and PREC + N50. These results suggested that precipitation reduction promoted plant-derived C accumulation through increasing aggregate stability; in contrast, under the N50 and PREC + N50, SOC increases through microbial residue accumulation. • Pathways of SOM formation under N deposition and rainfall decrease were clarified. • Rainfall reduction stimulated SOM accumulation by increasing aggregate stability. • Microbial residue accumulation enhanced SOM stability under N deposition. • The 13C natural abundance distinguished C flows in response to climate change. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) add a further layer of complexity to the proteome and regulate a wide range of cellular protein functions. With the increasing number of known PTM sites, it becomes imperative to understand their functional interplays. In this study, we proposed a novel analytical strategy to explore functional relationships between PTM sites by testing their tendency to be modified together (co-occurrence) under the same condition, and applied it to proteome-wide human phosphorylation data collected under 88 different laboratory or physiological conditions. Co-occurring phosphorylation occurs significantly more frequently than randomly expected and include many known examples of cross-talk or functional connections. Such pairs, either within the same phosphoprotein or between interacting partners, are more likely to be in sequence or structural proximity, be phosphorylated by the same kinases, participate in similar biological processes, and show residue co-evolution across vertebrates. In addition, we also found that their co-occurrence states tend to be conserved in orthologous phosphosites in the mouse proteome. Together, our results support that the co-occurring phosphorylation are functionally associated. Comparison with existing methods further suggests that co-occurrence analysis can be a useful complement to uncover novel functional associations between PTM sites. [ABSTRACT FROM AUTHOR]

Here, we report an unconventional Chinese pedigree consisting of three branches all segregating prelingual hearing loss (HL) with unclear inheritance pattern. After identifying the cause of one branch as maternally inherited aminoglycoside-induced HL, targeted next generation sequencing (NGS) was applied to identify the genetic causes for the other two branches. One affected subject from each branch was subject to targeted NGS whose genomic DNA was enriched either by whole-exome capture (Agilent SureSelect All Exon 50 Mb) or by candidate genes capture (Agilent SureSelect custom kit). By NGS analysis, we identified that patients from Branch A were compound heterozygous for p.E1006K and p.D1663V in the CDH23 (DFNB12) gene; and patients from Branch B were homozygous for IVS7-2A>G in the SLC26A4 (DFNB4) gene. Both CDH23 mutations altered conserved calcium binding sites of the extracellular cadherin domains. The co-occurrence of three different genetic causes in this family was exceedingly rare but fully compatible with the mutation spectrum of HL. Our study has also raised several technical and analytical issues when applying the NGS technique to genetic testing. [ABSTRACT FROM AUTHOR]

Autosomal dominant types of nonsyndromic hearing loss (ADNSHL) are typically postlingual in onset and progressive. High genetic heterogeneity, late onset age, and possible confounding due to nongenetic factors hinder the timely molecular diagnoses for most patients. In this study, exome sequencing was applied to investigate a large Chinese family segregating ADNSHL in which we initially failed to find strong evidence of linkage to any locus by whole-genome linkage analysis. Two affected family members were selected for sequencing. We identified two novel mutations disrupting known ADNSHL genes and shared by the sequenced samples: c.328C>A in COCH (DFNA9) resulting in a p.Q110K substitution and a deletion c. 2814_2815delAA in MYO6 (DFNA22) causing a frameshift alteration p.R939Tfs*2. The pathogenicity of novel coding variants in ADNSHL genes was carefully evaluated by analysis of co-segregation with phenotype in the pedigree and in light of established genotype-phenotype correlations. The frameshift deletion in MYO6 was confirmed as the causative variant for this pedigree, whereas the missense mutation in COCH had no clinical significance. The results allowed us to retrospectively identify the phenocopy in one patient that contributed to the negative finding in the linkage scan. Our clinical data also supported the emerging genotype-phenotype correlation for DFNA22. [ABSTRACT FROM AUTHOR]

We describe a large four-generational Chinese pedigree segregating MYH9-related disease caused by a V1516L mutation. The clinical findings supported previously established genotype-phenotype correlations, and also demonstrated interindividual variability of disease manifestations even within the same family. The same mutation was previously reported in another Chinese pedigree but resulting from a different DNA substitution. Analyzing the patterns of previously reported mutations revealed a limited spectrum of pathogenic variants. The implications of this finding are discussed. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10-8), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH. [ABSTRACT FROM AUTHOR]

Host genetic factors play an important role in diverse host outcomes after influenza A (H7N9) infection. Studying differential responses of inbred mouse lines with distinct genetic backgrounds to influenza virus infection could substantially increase our understanding of the contributory roles of host genetic factors to disease severity. Here, we utilized an integrated approach of mRNA-seq and miRNA-seq to investigate the transcriptome expression and regulation of host genes in C57BL/6J and DBA/2J mouse strains during influenza virus infection. The differential pathogenicity of influenza virus in C57BL/6J and DBA/2J has been fully demonstrated through immunohistochemical staining, histopathological analyses, and viral replication assessment. A transcriptional molecular signature correlates to differential host response to infection has been uncovered. With the introduction of temporal expression pattern analysis, we demonstrated that host factors responsible for influenza virus replication and host-virus interaction were significantly enriched in genes exhibiting distinct temporal dynamics between different inbred mouse lines. A combination of time-series expression analysis and temporal expression pattern analysis has provided a list of promising candidate genes for future studies. An integrated miRNA regulatory network from both mRNA-seq and miRNA-seq revealed several regulatory modules responsible for regulating host susceptibilities and disease severity. Overall, a comprehensive framework for analyzing host susceptibilities to influenza infection was established by integrating mRNA-seq and miRNA-seq data of inbred mouse lines. This work suggests novel putative molecular targets for therapeutic interventions in seasonal and pandemic influenza. [ABSTRACT FROM AUTHOR]

• Detection of soil aggregate stability related to soil organic carbon stability under N addition. • Fine roots play a major role in decreasing soil aggregate stability in response to N addition. • N addition enhances SOC stability with evidence of the decrease in C mineralization. • Stable mixed model revealed the promotion of mineral-associated C formation under N addition. • SOC stability in large macroaggregates was more vulnerable than that in small macroaggregates. Soil aggregates and the stability of their associated soil organic carbon (SOC) are important factors mediating soil carbon (C) sequestration and soil functions. However, the response of SOC stability to nitrogen (N) deposition is highly divergent, and the combined influences of N deposition and soil aggregates on SOC stability are poorly understood. The mechanisms underlying these influences were explored in a six-year field N -addition experiment covering a wide range of soil aggregates, root morphologies, soil properties and several SOC stability indices (represented by heterotrophic respiration and δ13C or δ15N) in a deciduous broad-leaved forest in Northeast China. The results showed that N addition significantly decreased the proportion of large macroaggregates (2–8 mm), reducing the soil aggregate mean weight diameter (MWD) by 10.0–17.2 %, which was negatively correlated with root length density (RLD) and root weight density (RWD). Organic C stability in soil aggregates was enhanced by N addition, as indicated by the decrease in the decomposition rate of organic C and the increase in the δ13C values but not the C content in microaggregates within macroaggregates (mM) or δ15N values. Furthermore, the promotion of mineral-associated organic C formation after N addition was detected by stable isotopic mixed model analysis (SIMM), indicating increased C protection by minerals. With the use of a structural equation model (SEM), the variation in the C stability of large macroaggregates (2–8 mm) was explained by the changes in fine roots, MWD and N availability but not those in small macroaggregates (0.25–2 mm) due to the instability of large macroaggregates. These results demonstrate that N addition may enhance soil C stability in all the soil aggregate sizes by promoting mineral sorption of C, with the C stability in large macroaggregates being more vulnerable to multiple environmental changes than that in small macroaggregates. Therefore, the soil C stability response to N deposition in the temperate forests of Northeast China could be modulated by soil aggregate sizes and may cause negative feedbacks to global warming. [ABSTRACT FROM AUTHOR]

Background and aim: Global nitrogen (N) deposition has been proposed to enhance phosphorus (P) limitation in various terrestrial ecosystems. The impact of N addition on soil P transformation, considering both microbial and abiotic properties, is not well understood.In this study, the experiment with three levels of N addition (0 (N0, no fertilizer), 25 (N25) and 50 kg N ha−1 yr−1 (N50)) was implemented in a temperate broad-leaved forest to assess the long-term (12 years) effects of N addition on soil P fractions associated with soil properties, iron, aluminum, calcium, phospholipid fatty acids (PLFAs), and enzyme activities.The results indicated a significant decrease in labile P, despite of a significant increase of approximately 54.0% in available P under N addition (N50). In contrast, the moderately labile P significantly increased under N addition treatment because of the increase in organic P in less labile fractions. The redundancy analysis and mantel-test found soil pH and MBP contributed to the variation of soil P fractions. The results of structural equation model confirmed that the microbial biomass P play a key role in the transformation of soil available P into moderately and occluded P fractions.These results suggested that the long-term addition of N decreased soil labile P and increased moderate and occluded P fractions through increasing microbial P use efficiency with increased MBP, leading to the enhancement of soil P limitation in the broad-leaved temperate forest.Methods: Global nitrogen (N) deposition has been proposed to enhance phosphorus (P) limitation in various terrestrial ecosystems. The impact of N addition on soil P transformation, considering both microbial and abiotic properties, is not well understood.In this study, the experiment with three levels of N addition (0 (N0, no fertilizer), 25 (N25) and 50 kg N ha−1 yr−1 (N50)) was implemented in a temperate broad-leaved forest to assess the long-term (12 years) effects of N addition on soil P fractions associated with soil properties, iron, aluminum, calcium, phospholipid fatty acids (PLFAs), and enzyme activities.The results indicated a significant decrease in labile P, despite of a significant increase of approximately 54.0% in available P under N addition (N50). In contrast, the moderately labile P significantly increased under N addition treatment because of the increase in organic P in less labile fractions. The redundancy analysis and mantel-test found soil pH and MBP contributed to the variation of soil P fractions. The results of structural equation model confirmed that the microbial biomass P play a key role in the transformation of soil available P into moderately and occluded P fractions.These results suggested that the long-term addition of N decreased soil labile P and increased moderate and occluded P fractions through increasing microbial P use efficiency with increased MBP, leading to the enhancement of soil P limitation in the broad-leaved temperate forest.Results: Global nitrogen (N) deposition has been proposed to enhance phosphorus (P) limitation in various terrestrial ecosystems. The impact of N addition on soil P transformation, considering both microbial and abiotic properties, is not well understood.In this study, the experiment with three levels of N addition (0 (N0, no fertilizer), 25 (N25) and 50 kg N ha−1 yr−1 (N50)) was implemented in a temperate broad-leaved forest to assess the long-term (12 years) effects of N addition on soil P fractions associated with soil properties, iron, aluminum, calcium, phospholipid fatty acids (PLFAs), and enzyme activities.The results indicated a significant decrease in labile P, despite of a significant increase of approximately 54.0% in available P under N addition (N50). In contrast, the moderately labile P significantly increased under N addition treatment because of the increase in organic P in less labile fractions. The redundancy analysis and mantel-test found soil pH and MBP contributed to the variation of soil P fractions. The results of structural equation model confirmed that the microbial biomass P play a key role in the transformation of soil available P into moderately and occluded P fractions.These results suggested that the long-term addition of N decreased soil labile P and increased moderate and occluded P fractions through increasing microbial P use efficiency with increased MBP, leading to the enhancement of soil P limitation in the broad-leaved temperate forest.Conclusion: Global nitrogen (N) deposition has been proposed to enhance phosphorus (P) limitation in various terrestrial ecosystems. The impact of N addition on soil P transformation, considering both microbial and abiotic properties, is not well understood.In this study, the experiment with three levels of N addition (0 (N0, no fertilizer), 25 (N25) and 50 kg N ha−1 yr−1 (N50)) was implemented in a temperate broad-leaved forest to assess the long-term (12 years) effects of N addition on soil P fractions associated with soil properties, iron, aluminum, calcium, phospholipid fatty acids (PLFAs), and enzyme activities.The results indicated a significant decrease in labile P, despite of a significant increase of approximately 54.0% in available P under N addition (N50). In contrast, the moderately labile P significantly increased under N addition treatment because of the increase in organic P in less labile fractions. The redundancy analysis and mantel-test found soil pH and MBP contributed to the variation of soil P fractions. The results of structural equation model confirmed that the microbial biomass P play a key role in the transformation of soil available P into moderately and occluded P fractions.These results suggested that the long-term addition of N decreased soil labile P and increased moderate and occluded P fractions through increasing microbial P use efficiency with increased MBP, leading to the enhancement of soil P limitation in the broad-leaved temperate forest. [ABSTRACT FROM AUTHOR]

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies. [ABSTRACT FROM AUTHOR]

Background: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by pathogenic remodeling of pulmonary arterioles leading to increased pulmonary pressures, right ventricular hypertrophy, and heart failure. PAH can be associated with other diseases (APAH: connective tissue diseases, congenital heart disease, and others) but often the etiology is idiopathic (IPAH). Mutations in bone morphogenetic protein receptor 2 (BMPR2) are the cause of most heritable cases but the vast majority of other cases are genetically undefined. Methods: To identify new risk genes, we utilized an international consortium of 4241 PAH cases with exome or genome sequencing data from the National Biological Sample and Data Repository for PAH, Columbia University Irving Medical Center, and the UK NIHR BioResource – Rare Diseases Study. The strength of this combined cohort is a doubling of the number of IPAH cases compared to either national cohort alone. We identified protein-coding variants and performed rare variant association analyses in unrelated participants of European ancestry, including 1647 IPAH cases and 18,819 controls. We also analyzed de novo variants in 124 pediatric trios enriched for IPAH and APAH-CHD. Results: Seven genes with rare deleterious variants were associated with IPAH with false discovery rate smaller than 0.1: three known genes (BMPR2, GDF2, and TBX4), two recently identified candidate genes (SOX17, KDR), and two new candidate genes (fibulin 2, FBLN2; platelet-derived growth factor D, PDGFD). The new genes were identified based solely on rare deleterious missense variants, a variant type that could not be adequately assessed in either cohort alone. The candidate genes exhibit expression patterns in lung and heart similar to that of known PAH risk genes, and most variants occur in conserved protein domains. For pediatric PAH, predicted deleterious de novo variants exhibited a significant burden compared to the background mutation rate (2.45×, p = 2.5e−5). At least eight novel pediatric candidate genes carrying de novo variants have plausible roles in lung/heart development. Conclusions: Rare variant analysis of a large international consortium identified two new candidate genes—FBLN2 and PDGFD. The new genes have known functions in vasculogenesis and remodeling. Trio analysis predicted that ~ 15% of pediatric IPAH may be explained by de novo variants. [ABSTRACT FROM AUTHOR]

Background: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by pathogenic remodeling of pulmonary arterioles leading to increased pulmonary pressures, right ventricular hypertrophy, and heart failure. PAH can be associated with other diseases (APAH: connective tissue diseases, congenital heart disease, and others) but often the etiology is idiopathic (IPAH). Mutations in bone morphogenetic protein receptor 2 (BMPR2) are the cause of most heritable cases but the vast majority of other cases are genetically undefined. Methods: To identify new risk genes, we utilized an international consortium of 4241 PAH cases with exome or genome sequencing data from the National Biological Sample and Data Repository for PAH, Columbia University Irving Medical Center, and the UK NIHR BioResource – Rare Diseases Study. The strength of this combined cohort is a doubling of the number of IPAH cases compared to either national cohort alone. We identified protein-coding variants and performed rare variant association analyses in unrelated participants of European ancestry, including 1647 IPAH cases and 18,819 controls. We also analyzed de novo variants in 124 pediatric trios enriched for IPAH and APAH-CHD. Results: Seven genes with rare deleterious variants were associated with IPAH with false discovery rate smaller than 0.1: three known genes (BMPR2, GDF2, and TBX4), two recently identified candidate genes (SOX17, KDR), and two new candidate genes (fibulin 2, FBLN2; platelet-derived growth factor D, PDGFD). The new genes were identified based solely on rare deleterious missense variants, a variant type that could not be adequately assessed in either cohort alone. The candidate genes exhibit expression patterns in lung and heart similar to that of known PAH risk genes, and most variants occur in conserved protein domains. For pediatric PAH, predicted deleterious de novo variants exhibited a significant burden compared to the background mutation rate (2.45×, p = 2.5e−5). At least eight novel pediatric candidate genes carrying de novo variants have plausible roles in lung/heart development. Conclusions: Rare variant analysis of a large international consortium identified two new candidate genes—FBLN2 and PDGFD. The new genes have known functions in vasculogenesis and remodeling. Trio analysis predicted that ~ 15% of pediatric IPAH may be explained by de novo variants. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: The 5′ HoxD genes and their paralogs in the HoxD cluster are crucial for normal vertebrate limb development. Mutations in HOXD13 and HOXD13 have been found to cause human limb malformation. Here we describe a two-generation Chinese family with a variant form of mild synpolydactyly. Methods: Sequence analysis of HOXD13 gene in a two-generation Chinese family with six individuals. Results: Gene scan and linkage analysis suggested that HOXD13 might be responsible for the disease of this family. An LOD around 1.8 was observed at three markers (P =2E−3). We identified a novel c.893G>A (p.Arg298Gln) mutation in the HOXD13 homeodomain. And the mutation affected the transcriptional activation ability of HOXD13. Conclusion: This finding expands the phenotypic spectrum associated with HOXD13 mutations and advances our understanding of human limb development. [Copyright &y& Elsevier]

Abstract: Vertebrate genomes encode thousands of non-coding RNAs including short non-coding RNAs (such as microRNAs) and long non-coding RNAs (lncRNAs). Chicken (Gallus gallus) is an important model organism for developmental biology, and the recently assembled genome sequences for chicken will facilitate the understanding of the functional roles of non-coding RNA genes during development. The present study concerns the first systematic identification of lncRNAs using RNA-Seq to sample the transcriptome during chicken muscle development. A computational approach was used to identify 281 new intergenic lncRNAs in the chicken genome. Novel lncRNAs in general are less conserved than protein-coding genes and slightly more conserved than random non-coding sequences. The present study has provided an initial chicken lncRNA catalog and greatly increased the number of chicken ncRNAs in the non-protein coding RNA database. Furthermore, the computational pipeline presented in the current work will be useful for characterizing lncRNAs obtained from deep sequencing data. [Copyright &y& Elsevier]

Awnness is a key trait in rice domestication, yet no studies have been conducted on fine mapping or association mapping of the rice awn gene. In this study, we investigated the awnness and genotype of a core collection of 303 cultivated rice varieties and a BC5F2 segregating population of 200 individuals. Combining association and linkage analyses, we mapped the awnness related genes to chromosome 4. Primary association analysis using 24 SSR markers revealed five loci significantly associated with awnness on chromosome 4. The associated markers cover previously identified regions. Fine association mapping was conducted using another 29 markers within a 4-Mb region, covering the associated marker in34, which is close to the awn gene Awn4.1. Seven associated markers were revealed, distributed over an 870-kb region. Combining the fine association mapping and linkage analysis of awnness in the 200 BC5F2 segregating population, we finally identified a 330-kb region as the candidate region for Awn4.1. The results indicate that combining association mapping and linkage mapping provides an efficient and precise approach to both genome-wide mapping and fine mapping of rice genes. [ABSTRACT FROM AUTHOR]

Na Zhu, Emilia M. Swietlik, Carrie L. Welch, Michael W. Pauciulo contributed equally to this work. Reference 1 Zhu N. Rare variant analysis of 4241 pulmonary arterial hypertension cases from an international consortium implicates FBLN2, PDGFD, and rare de novo variants in PAH. [Extracted from the article]

Alzheimer's disease (AD) is the most common neurodegenerative disorders in the elderly. To identify rare genetic factors other than apolipoprotein E ɛ4 allele (ApoE ɛ4) contributing to the pathogenesis of late-onset AD (LOAD), we conducted a whole-exome analysis of 246 ApoE ɛ4-negative LOAD cases and 172 matched controls in Hong Kong Chinese population. LOAD patients showed a significantly higher burden of rare loss-of-function variants in genes related to immune function than healthy controls. Among the genes involved in immune function, we identified a rare stop-gain variant (p.Q48X) in mixed lineage kinase domain like pseudokinase ( MLKL) gene present exclusively in 6 LOAD cases. MLKL is expressed in neurons, and the its expression levels in the p.Q48X carriers were significantly lower than that in age-matched wild-type controls. The ratio of Aβ42 to Aβ40 significantly increased in MLKL knockdown cells compared to scramble controls. MLKL loss-of-function mutation might contribute to late-onset ApoE ɛ4-negative AD in the Hong Kong Chinese population. [ABSTRACT FROM AUTHOR]