Pocillopora spp. exhibited an exceptional rapid increase from 22 to 51% relative benthic cover. By contrast, turf algae cover decreased from 63 to 24%, resulting in a corresponding increase in crustose coralline algae cover. The macroalga Caulerpa sertularioides covered up to 15% of the reef in April 2013, disappeared after synchronized gamete release in May, and subsequently exhibited slow regrowth. Parallel monitoring of influencing factors suggest that C. sertularioides cover was mainly regulated by their reproductive cycle, while that of turf algae was likely controlled by high abundances of herbivores. Upwelling events in February and March 2014 decreased mean daily seawater temperatures by up to 7 °C and increased nutrient concentrations up to 5- (phosphate) and 16-fold (nitrate) compared to mean values during the rest of the year. Changes in benthic community composition did not appear to correspond to the strong environmental changes, but rather shifted from turf algae to hard coral dominance over the entire year of observation. The exceptional high dynamic over the annual observation period encourages further research on the adaptation potential of coral reefs to environmental variability.Seasonal upwelling at the northern Pacific coast of Costa Rica offers the opportunity to investigate the effects of pronounced changes in key water parameters on fine-scale dynamics of local coral reef communities. This study monitored benthic community composition at Matapalo reef  by weekly observations of permanent benthic quadrats from April 2013 to April 2014. Monitoring was accompanied by surveys of herbivore abundance and biomass and measurements of water temperature and inorganic nutrient concentrations. Findings revealed that the reef-building corals  Coral reef benthic communities are controlled by abiotic and biotic drivers, such as waves, water depth, reef habitat, temperature, nutrients and herbivory . AnthropTo understand the effects of variable environmental drivers on coral reef ecosystems it is useful to monitor ecosystems with a pronounced natural temporal variability in conditions, such as upwelling regions. Transport of subthermocline water to the surface  and internal bores) acts to decrease sea water temperature and increase nutrient concentrations in reef waters on different temporal scales ranging from minutes to several days . PreviouWe selected the Gulf of Papagayo at the northern Pacific coast of Costa Rica to investigate the question of how annual variability in environmental parameters affects coral reef communities. A topographic depression in the lowlands of southern Nicaragua and northern Costa Rica allows strong winds to blow across from the Caribbean during the northern hemisphere winter . On the The present study monitored the temporal variation in a reef community exposed to moderate upwelling in a weekly resolution over one year. Observations of benthic community composition were accompanied by monitoring of herbivores (sea urchin abundance and fish biomass) and key water parameters (temperature and inorganic nutrient concentrations). The objectives of the study were to (1) observe the dynamics in benthic community composition over one year, (2) identify the influencing biological and (3) environmental factors and (4) develop an ecological perspective for local coral reefs. Important related key questions were: (i) which benthic groups benefit from seasonal changes in biotic and abiotic parameters? (ii) which parameter has the strongest effect on benthic community composition? and (iii) does the benthic community follow a seasonal cycle?Pocillopora and stretches 1.2 km from Punta Matapalito to the west end of Playa Matapalo , 500 m north–west of Playa Blanca (study site in n = 5) were permanently marked with iron stakes and repeatedly observed for changes in benthic coverage every week. The location of permanent quadrats was chosen by placing a 50 × 50 cm frame alternately left and right every 2 m along a transect line parallel to the coast in 5 m water depth. For observation, the PVC frame with a 5 × 5 cm grid was placed over the stakes as a reference, and live coral, dead coral, crustose coralline algal (CCA), turf algal, macroalgal, sand, rubble and sessile benthic invertebrate cover on the substrate was quantified from directly above the grid using SCUBA. The weekly quantification was done on a relatively small area of the reef, but results were supported by monthly chain surveys along transects covering the whole reef patch. Transects of 10 m length (n = 5) were permanently marked with iron stakes parallel to the coast in 5 m water depth in about 3 m distance to the permanent quadrats. A 10 m iron chain with 532 links was laid out over the reef substrate along each transect, and seafloor coverage was observed under each link. Proportional seafloor cover was calculated by relating the average number of links in each category to the total number of links, and rugosity was calculated by dividing the horizontal distance covered by the chain (d) by its real length (l) (rugosity index: RI = 1 − d/l).Monitoring took place on a reef patch of around 600 mDiadema mexicanum and Eucidaris thourasii were counted in 1 m belts on both sides of the transect lines  from April 2013 to April 2014. Fish surveys were conducted from November 2013 to March 2014. During surveys, all individuals (excluding cryptic species) within 2.5 m belts on both sides of the transect lines  were identified to species level and assigned to size classes . Multiple swims (n = 3) with intervals of 5 min between swims were conducted on each transect in order to increase the precision of data for each transect. Fish species were categorized into trophic groups according to their dominant food source  according to descriptions on FishBase (H) and Evenness (HE) was calculated for each survey day from the number and relative contribution of each fish species to total fish abundance counted on all 5 transects.Surveys of sea urchin and fish abundances were conducted monthly between 9:00 and 14:00 on all five permanent transects using SCUBA. Sea urchins of the species FishBase . Biomass® Laboratory Fluorometer/Photometer (Turner Designs) according to −1). Determination of phosphate (PO−1). Sub-samples were kept dark and frozen until the end of the study period and were analyzed for nitrate (NO® photometer based on a method revised by x)(NO = 0.162 µmol L−1). A detailed description of the temporal variability in physicochemical and organic parameters at the study site can be found in Monitoring of benthic community composition was accompanied by measurements of temperature with a Manta 2 Water Quality Multiprobe deployed directly on the reef substrate  and weekly determination of inorganic nutrient concentrations. Water samples for nutrient measurements were taken in triplicate from directly above the reef surface within glass jars (500 mL), filtered immediately through disposable syringe filters (pore size 0.45 µm) and stored cool for transportation. Ammonia Pocillopora spp., CCA, C. sertularioides, turf, sand, rubble, cyanobacteria) and between sea urchin abundances and turf algal cover were tested via Pearson Product Moment Correlation in SigmaPlot 13. Temporal dynamics in environmental parameters and benthic community composition were examined with Principal Coordinate Ordinations (PCO)  was calculated using Bray Curtis similarity. The fixed factors used for the analysis were (1) upwelling period with two levels  and (2) individual months with 13 levels (each month from April 2013 to April 2014). To test how well patterns in community composition correlated to patterns in environmental parameters, the function RELATE in PRIMER 6 was used, which tests matching of the resemblance matrices by Spearman Rank correlation. Values in the text are reported as mean ± standard error (SE) if not stated otherwise.Differences in fish and sea urchin abundances over time were tested with one way ANOVAs followed by Tukey post-hoc tests in SigmaPlot13 for Windows. Correlations between cover types (ns (PCO)  and testns (PCO)  in PRIMEPocillopora spp. (P. elegans and P. damicornis), crustose coralline algae (CCA) and turf algae. Turf algae communities existed as fine tufts of filamentous algae and cyanobacteria or in denser conglomerates with Dictyota spp. The green algae Caulerpa sertularioides was the only individually growing fleshy macroalga on the reef and covered large parts of the dead reef framework in shallow areas and on the edges of reef patches. Single coral colonies of Pocillopora spp., Pavona spp. and Psammocora spp. and coral rubble could be found on sandy patches between the reef rock areas. The rugosity of the reef substrate was low with values between 0.08 and 0.16.At Matapalo reef, areas of dead reef structure overgrown with turf algae alternate with healthy reef areas dominated by live coral cover. At the time of study, the benthic community in healthy reef areas was dominated by the sole reef building corals Pocillopora spp. increased continuously from 22% in April 2013 to 51% in April 2014. Turf algae initially covered around 60% of the substrate, but decreased to 35% within two weeks in June 2013. The drop in turf algae cover resulted in an increase in the relative cover of CCA from 15 to 30%, as previously turf algae had overgrown much of the CCA-covered substrate. CCA cover remained around 30% until January 2014, when it decreased slightly coincident with an increase in coral cover. The macroalga Caulerpa sertularioides initially covered 3% of the substrate in the permanent quadrats in April 2013, but decreased to 0.1–0.5% by May 2013. Macroalgae subsequently exhibited temporary increases in cover of up to 5% in August 2013 and 2.5% in March 2014. Percent cover of Pocillopora spp. and turf algae in permanent quadrats correlated significantly to their respective coverages in the monthly chain transects covering the whole reef patch . By contrast, correlations of CCA and C. sertularioides coverages between the quadrats and chain transects were not significant , due to the fact that the cover of C. sertularioides was higher towards the peripheries of the reef patch. The drop in C. sertularioides cover from April to May 2013 could therefore be observed in a more pronounced way in the chain transects across the whole reef patch . Cover of turf algae in permanent quadrats correlated negatively with that of Pocillopora spp. , CCA (r = − 0.613) sand (r = − 0.674) and rubble . Sand and rubble also correlated positively with each other (r = 0.834) and with the cover of Pocillopora spp.  and cyanobacteria .Weekly monitoring of permanent quadrats revealed a major shift from turf algae to hard coral dominance within the year of observation . Cover oPocillopora spp., and negatively with turf algae. From October 2013 to April 2014, the composition shifted further along PCO2, which explained 17.4% of the total variance and showed a positive correlation to rubble and a negative correlation to CCA. Differences in community composition were significant between months  = 47.848, P(perm) = 0.001, perms = 998).Multivariate analyses further illustrated the pronounced shift in benthic community composition over the study period . From Ap−2 for Diadema mexicanum and 0.16 ± 0.03 ind. m−2 for the pencil sea urchin Eucidaris thourasii  = 0.991, P = 0.470 and F = 1.336, P = 0.228, respectively). Abundances of D. mexicanum correlated positively with turf algae cover in permanent quadrats  and on benthic transects .Sea urchin abundances in the reef averaged 5.31 ± 0.36 ind. mhourasii . IndividH = 1.7 in March 2014 to H = 2.3 in November 2013 and was positively related to the evenness of species distribution (HE = 0.59–0.78). Small groupers were the only predators recorded during fish surveys at Matapalo reef. Invertebrate feeders  dominated total abundance and biomass of fish except in December 2013, when high abundances of planktivorous Chromis atrilobata dominated fish counts, and in March 2014, when many large individuals of the herbivorous parrotfish Scarus ghobban were present  = 6.095, P = 0.002) and total biomass was significantly elevated in November and December 2013  = 14.729, P < 0.001). A statistical correlation of fish abundance or biomass with benthic community composition was not conducted due to the low sample size of fish data (n = 5).The Shannon–Wiener diversity of the observed fish community ranged from  present  and 4D. Short drops in temperature of approximately 2 °C occurred at Matapalo reef in June, October and December 2013. More pronounced and longer lasting cooling events, coupled with increased nutrient concentrations, occurred between February and March 2014  and 5B. r = − 0.86), r = 0.93), r = 0.83) and r = 0.95)  = 19.87, P(perm) = 0.001, perms = 999) and among individual months  = 2.7984, P(perm) = 0.006, perms = 997). The temporal pattern in environmental parameters did not correlate to the pattern in community composition  = 0.332, perms = 999) and therefore did not explain any variance in the benthic community data.According to the PCO analysis, the variability in environmental parameters at Matapalo from April 2013 to April 2014 was mainly explained along PCO1 , which highly correlated with temperature ( = 0.95) . EnvironThis is the first study to describe the variability in a benthic coral reef community in response to both an upwelling period and fluctuations in herbivore abundance in a high temporal resolution. The observations demonstrate the dynamic nature of the benthic community at the study site and provide detailed baselines for benthic community composition as well as fish and sea urchin abundances in coral reefs of the eastern tropical Pacific.Lobophora variegata (Acropora (up to 100–200% within 6–12 months) to fast tissue regeneration of small remnants of unbleached live tissue, the high competitive capacity of the corals, and a seasonal dieback in the blooming seaweed. Similarly, the increase in coral cover during the present study did not involve new coral recruitment  at our study site suggest they may provide a key control of algal abundance also in the eastern tropical Pacific. We suggest that grazing by sea urchins was especially important in reducing turf algae abundance after a bloom at the beginning of the study period and was therefore responsible for the pronounced decrease in turf algae cover observed in June 2013. Abundances of D. mexicanum were positively correlated with turf algae cover, indicating migration or recruitment patterns of sea urchins according to food availability. By removing turf algae, D. mexicanum uncovered underlying crustose coralline algae and thereby likely facilitated the observed increase in live coral cover. High abundances of Diadema spp. can however also damage a reef by extensively eroding the carbonate substrate  was comparable to baselines established in the south–western Atlantic  was lower than mean values for the Indo-Pacific, but higher than in the Caribbean  compromised most of the herbivorous fish biomass and was similar to mean values for the Indo-Pacific but much higher than those for the Caribbean (13.1 ± 2.4 and 6.7 ± 0.7 g m−2, respectively; Total reef fish biomass at Matapalo (51 ± 13 g mA parallel manipulative study at Matapalo reef showed that herbivorous fish controlled turf and fleshy macroalgal growth on long-term exposed terracotta settlement tiles, even during the nutrient-rich upwelling period . In contTurf algae harbor high abundances of microbes, including potential coral pathogens, and may therefore increase the prevalence of coral diseases and further negatively impact coral health . The preThe continuous increase in live coral cover over the studied year suggests that the investigated reef patch was recovering from a disturbance event in the past. However, the increase in coral cover was not apparent for all parts of the 1.2 km long reef. Furthermore, rapidly increasing coral cover does not always indicate a healthy and resilient reef . RepeateC. sertularioides appeared to be controlled by inorganic nutrient availability. Contrary to initial expectations, the benthic community composition did not follow a seasonal cycle corresponding to the non-upwelling and upwelling period, but instead shifted from turf algae to coral dominance over the year of observation. Interannual variability in conditions and community responses are likely high and future studies should address the hypothesis that local reef communities are well adapted to upwelling conditions. The observation that sea urchins largely controlled turf algae cover in the reef should be verified via exclusion experiments.In conclusion, hard corals and CCA benefited from prevailing environmental conditions during the year of observation, while cover of turf algae and fleshy macroalgae declined. Herbivores likely controlled turf algae growth, thereby facilitating fast coral expansion. In contrast, cover of the macroalga 10.7717/peerj.1434/supp-1Supplemental Information 1Click here for additional data file.


Poly[A]+ RNA-Seq analyses of normal human adult tissue samples such as Illumina’s Human BodyMap 2.0 Project and the RNA-Seq atlas have provided a useful global resource and framework for comparisons with diseased tissues such as cancer. However, these analyses have failed to provide information on poly[A]−RNA, which is abundant in our cells. The most recent advances in RNA-Seq analyses use ribosomal RNA-depletion to provide information on both poly[A]+ and poly[A]−RNA. In this paper, we describe the use of Illumina’s HiSeq 2000 to generate high quality rRNA-depleted RNA-Seq datasets from human fetal and adult tissues. The datasets reported here will be useful in understanding the different expression profiles in different tissues.Gene expression is the most fundamental level at which the genotype leads to the phenotype of the organism. Enabled by ultra-high-throughput next-generation DNA sequencing, RNA-Seq involves shotgun sequencing of fragmented RNA transcripts by next-generation sequencing followed by  Next Generation Sequencing (NGS) technique uses short read lengths to enable the massive parallel sequencing of the genome. Currently with paired-end sequencing and multiplexing technology, NGS allows multiple samples to be studied at the same time, dramatically reducing the time spent generating data and simplifying the procedure. RNA-Seq is a technique used to study the transcriptome and involves the direct sequencing of transcripts by Next Generation Sequencing Technologies . It alloThere are various different databases on the web which provide information about the expression profiles of different tissues using RNA-Seq. Some of these databases include Illumina’s Human BodyMap 2.0 and the RNA-Seq Atlas. Illumina’s Human BodyMap 2.0 project data was generated in 2010 using its HiSeq 2000 instrument. This project consisted of 16 adult human tissue types including adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid, and white blood cells from different individuals. RNA-Seq libraries were constructed using poly[A]+ mRNA and sequenced using small RNA-sequencing. For each tissue, the raw reads were aligned to the genome and then the exons were linked into tissue-specific transcript models using the reads that span an exon-exon boundary. A diverse RNA-Seq library database of 16 different tissues is available which makes it very comprehensive. However, there is no information about poly[A]− RNA.1. However, this database lacks the information of fetal tissues as only adult tissues are included in this study. Hence, our study aims to address this situation and allow the comparison between adult and fetal tissues2.The RNA-Seq Atlas data was generated from 11 healthy, human tissue samples pooled from multiple donors. The tissues include adipose, colon, heart, hypothalamus, kidney, liver, lung, ovary, skeletal muscle, spleen and testes. Both poly[A]+ and poly[A]− RNA were used for sequencing. The expression levels were estimated by mapping and counting reads to single gene sequences derived from the UCSC genome browser, followed by normalization to Reads Per Kilobase of exon model per Million (RPKM) mapped reads values3. The use of rRNA-depleted RNA-Seq on mouse cerebrum tissues has enabled more novel transcribed loci to be detected than mRNA-seq with the former yielding 9,428 novel transcribed loci and the latter yielding only 4,550 , providing more information on splicing patterns and splice junctions4. Ribosomal depleted RNA datasets such as the characterization of the landscape of transcription in humans as part of the ENCODE consortium has allowed the further annotation of the human genome and discovery of novel transcripts5 and recently has been used to identify zero nucleotide recursive splicing in drosophila and humans6.Ribosomal RNA (rRNA) is the most abundant transcript in total RNA and constitutes about 80% of the total RNA, while poly[A]+ mRNA constitutes only about 5% of the total RNA present in an eukaryotic cell. rRNA provides little information about the transcriptome, hence it would be beneficial to remove rRNA to maximise the amount of information retrieved from a sequencing run. The use of ribosomal depleted RNA has been shown to recover more information about protein-coding genes, non-coding RNAs, snRNAs, snoRNAs and repeat elementsOur project aims to compare the expression between different fetal and adult tissues and to identify tissue-specific genes and expression profiles. Constructing ribosomal depleted RNA-Seq libraries in fetal tissues would provide insights into the early gene expression patterns in these tissues. Studying both fetal and adult gene expressions could give us a greater understanding of the regulated genes in different developmental stages and its effects on developmental functions. Study of these RNA-sequences will allow us to determine tissue-specific splice variants as well as non-coding RNA. Genome wide screens in different tissue types might also help us to identify polymorphisms and their effects on overall expression and splicing.2 different fetal normal tissues and 6 different adult normal tissues were purchased from different sources . The qualities of these total RNA were tested using the Agilent Bioanalyser 2100 Eukaryote Total RNA Nano Series II. Only total RNAs with a RIN score of more than 7 were used for RNA-Seq library construction. Details of the total RNAs used were listed in Ribosomal RNA (rRNA) was removed from total RNA using the RiboMinus Eukaryote Kit for RNA-Seq from Ambion. The ribosomal RNA depleted RNA fraction is termed the RiboMinus RNA fraction and is enriched in polyadenylated (polyA) mRNA, non-polyadenylated RNA, pre-processed RNA, tRNA, and may also contain regulatory RNA molecules such as microRNA (miRNA) and short interfering RNA (siRNA), snRNA, and other RNA transcripts of yet unknown function. Ambion RiboMinus rRNA depletion was performed as described in the manufacturer’s protocol  following the standard protocol.TruSeq RNA Sample Preparation was performed on the RiboMinus RNA fraction as described in the manufacturer’s protocol  following the low sample protocol. The libraries were sequenced on Illumina’s HiSeq 2000 instrument.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), picard tools (http://picard.sourceforge.net) RseQC7. The reads were aligned to the UCSC human genome release version hg19 reference genome using STAR8 and annotated using the Gencode version 19 annotation. Subsequent quantification of expression levels and isoforms was done using RSEM9. The STAR_RSEM.sh script was used for the processing of the data (https://github.com/ENCODE-DCC/long-rna-seq-pipeline/blob/master/DAC/STAR_RSEM.sh). The aligned reads were uploaded into ZENBU10 a genome browser for visualization. The program featureCounts11 was used to summarize the features of all the RNA-seq library. The output from featureCounts was inputted into the program edgeR12 to generate the multidimensional scaling plot.The quality of the RNA-seq libraries were evaluated using fastQC (The raw fastq files for the RNA-seq libraries were deposited at NCBI Sequence Read Archive (SRA) with BioProject accession PRJNA283012 (Data Citation 1: Sequence Read Archive PRJNA283012). The output from the quantification of transcripts by RSEM were deposited at Gene Expression Omnibus (GEO) with accession GSE69360 (Data Citation 2: Gene Expression Omnibus GSE69360). These files are tab-delimited files in the format suitable for viewing in the genome browser ZENBU. It contains the location of the transcripts on the genome, counts and normalized counts. The supplementary file GSE69360_RNAseq.counts.txt.gz contains counts used to construct the MDS plot.The integrity of the total RNA was measured by the RNA Integrity Number (RIN) algorithm; calculated by the Agilent Bioanalyzer software. It was determined by the entire electrophoretic trace of the RNA sample. This included the presence or absence of degradation products. The higher the RIN score, the better the integrity of the total RNA, with the highest RIN score of 10. All the total RNAs used for rRNA-depleted RNA-Seq had a RIN score of above 7 which showed the high integrity of total RNAs used. The Agilent Bioanalyzer profiles for the total RNA and RIN scores were listed in All the rRNA-depleted RNA-Seq libraries had a library size range between 250 to 400 bp which was similar to the expected library size distribution as shown in the TruSeq RNA Sample Preparation manufacturer’s protocol . This library size range was ideal for sequencing on the HiSeq 2000 instrument. The Agilent Bioanalyzer profiles for the rRNA-depleted RNA were listed in http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The average quality score per base per library for both the forward and reverse read was plotted. The average quality score per base was high with a median score above 30 indicating high quality sequences across all bases  with mitochondrial origin in some libraries particularly the colon, heart and kidney. This is consistent with the findings of the GTEx consortium that reported high levels of transcripts with mitochondrial origin from tissues with high aerobic activity such as the heart and kidneySeveral uses for the RNA-Seq libraries include identifying and validating new genes and transcripts and comparing expression in different fetal and adult tissues. In order to dissect out gene expression differences that arise due to different individuals, we profiled five normal stomachs from different people. Adult tissues which were used for this analysis included heart, lung, liver, kidney, stomach and colon. Fetal tissues included stomach and colon. In particular, the inclusion of liver and kidney will provide insights into drug toxicity, and the potential side effects of targeting particular drug targets. As normal samples, with no evidence of disease were used for these analyses, they are a useful complement to studies of cancer through RNA-Seq, which uses tissue adjacent to the cancer for comparison. While these tissues show no apparent cancer phenotype, they may not be completely normal, because cancer cells could infiltrate the adjacent tissues. In addition, the adjacent ‘normal’ cells could contain pre-malignant changes. Taken together, this dataset is a useful addition to the general biomedical community.How to cite this article: Choy, J. et al. A resource of ribosomal RNA-depleted RNA-Seq data from different normal adult and fetal human tissues. Sci. Data 2:150063 doi: 10.1038/sdata.2015.63 (2015).


Here, we discuss the current knowledge of m6A and m5C RNA methylation pathways and summarize how they impact normal tissues and contribute to human disease.Over the last five decades more than 100 types of RNA modifications have been identified in organism of all kingdoms of life, yet their function and biological relevance remain largely elusive. The recent development of transcriptome-wide techniques to detect RNA modifications such as N Current Opinion in Cell Biology 2014, 31:1–7Cell cycle, differentiation and diseaseThis review comes from a themed issue on Stefano Piccolo and Eduard BatlleEdited by Issue and the EditorialFor a complete overview see the Available online 10th July 2014http://dx.doi.org/10.1016/j.ceb.2014.06.006http://creativecommons.org/licenses/by/3.0/).0955-0674/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (5-methylcytidine (m5C) and N6-methyladenosine (m6A) has been described 50 years ago . T. T6A is quencing , 48••. mnd yeast . The conon sites . The occnditions .6A is thought to relate to various aspects of mRNA metabolism, including mRNA expression and degradation, splicing, translational regulation and regulation of microRNA-binding [6A regulating RNA-protein interactions, there is currently a considerable lack of evidence supporting other proposed functions in vivo. The presence of m6A in mRNA modulates the binding affinity to the RNA binding proteins Hu-antigen R (HUR) and YTHDF1–3, which in turn regulate the stability and cellular distribution of the bound mRNA [The addition of a single methyl group to adenosines does not perturb Watson–Crick base pairing, but it weakens RNA secondary structure . Thus, t-binding . Notablyund mRNA , 52, 53.6A in a large number of mRNAs, it is not surprising that this modification has been implicated in a wide range of cellular processes, and is likely to play an essential role in development and tissue differentiation by modulating cell fate and survival, stress responses, meiosis, the circadian clock, as wells as cellular immunity [Considering the high abundance of mimmunity , 55, 56.6A in mammals. The two methylases methyltransferase-like 3  and methyltransferase-like 14 (Mettl14) form the core of the complex and associate with additional regulatory factors such as WTAP (Wilm's tumour 1 associating protein)  c [52, 576A-methyltransferases are not fully understood but emerging evidence implicates a role in embryo development, gametogenesis and stem cell self-renewal. Mouse ES cells lacking Mettl3 and Mettl14 lost self-renewal capability and the decreased levels of m6A in mRNAs of developmental regulators correlated with binding of the mRNA stabilizer HUR, indicating that m6A methylation inversely correlated with mRNA stability and is needed to maintain pluripotency [The precise biological functions of mipotency . During ipotency . In adulipotency .5C-methylation, also RNA m6A-methylation can be reverted. Fat mass and obesity associated protein (Fto) and α-ketoglutarate-dependent dioxygenase alkB homolog 5 (AlkBH5) are demethylases that remove m6A from RNA (6A in RNA after Fto or AlkBH5 over-expression indicated substrate specificity and suggests the existence of additional demethylating enzymes [Similar to DNA mfrom RNA c [50••,  enzymes , 60.Genome-wide association studies linked common polymorphisms in the first intron of FTO to body mass index, risk of obesity, type 2 diabetes, polycystic ovary syndrome and cardiovascular diseases . StudiesIn addition, FTO polymorphisms in intron 1 have been linked to a range of human cancers, yet a recent meta-analysis study suggested that except for pancreatic cancer the increased cancer risk is rather associated to obesity than the FTO polymorphism itself . Other pWhile the functional consequences of single nucleotide polymorphisms in the intronic region of FTO are still unknown, loss-of-function mutations of FTO in humans lead to an autosomal-recessive lethal syndrome of severe growth retardation, microcephaly, psychomotor delay, cardiac deficits, and multiple malformations, and at least some of these effects may be due to impaired proliferation and accelerated senescence . SimilarThe only limited information available about AlkBH5 indicated an essential role in gametogenesis. AlkBH5 expression is highest in primary spermatocytes in the mouse testes, and its inactivation leads to testis atrophy and infertility due to failure to enter and proceed through spermatogenic differentiation .6A affects the fate of methylated mRNAs and lncRNAs. While some evidence suggests that m6A occurrence in mRNA is inversely correlated to stability [6A deposition plays essential roles in mRNA metabolism, and both m6A methylases and demethylases are crucial during embryonic development and homeostasis of the central nervous, cardiovascular and reproductive systems. Furthermore, aberrant m6A methylation pathways are linked to a range of human diseases including infertility, obesity as well as developmental and neurological disorders.In summary, it is not fully understood how mtability , it rema5C and m6A methylation, but a large number of other intriguing chemical modifications exist in RNAs. Thus, our current knowledge only scratches the surface of the many roles of post-transcriptional modifications in modulating transcriptional and translational processes.In only a couple years, our understanding about RNA methylation pathways advanced with remarkable speed and the importance of RNA methylation and its role in human diseases is now widely recognized. However, the precise molecular pathways and cellular processes regulated by these modifications are still largely unclear. Furthermore, we only described current advances on m5C in mRNA or other low abundant RNAs, second, map m6A at single nucleotide resolution and third, to identify other RNA modifications. To fully understand the biological roles of RNA methylation, it will be required to identify all RNA methylases, de-methylases, the regulatory pathways that control their activity and their specific RNA targets. A major goal will be to dissect the precise mechanisms how RNA modifications affect global and specific protein production. Indeed, a modest correlation between cellular mRNA and protein levels highlights the importance post-transcriptional and post-translational regulatory pathways. Ultimately, by understanding fundamental aspects of RNA modification biology we will be able to develop selective and specific small-molecule inhibitors to modulate RNA methylation levels. Such discoveries may well lead to the identification of novel therapeutic strategies to treat complex diseases including developmental and neurological disorders, obesity or cancer.Further advances in the field will rely on the development of new system-wide strategies to first, reliably detect m• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.  Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress. The HIF-α family members are dynamically regulated by a class of prolyl-hydroxylases (PHDs) and the Factor Inhibiting HIF (FIH); both of which require oxygen as a co-factor to catalyze the hydroxylation of HIF-α2,3. In normoxia the HIF-α family members are hydroxylated and tagged for proteosomal degradation by the E3-Ligase, von Hippel Lindau (vHL). In hypoxia the PHDs and FIH are inactive or have a reduced activity. The HIF-α isoforms become stabilized, form a heterodimer with HIF-1β, and effect the transcription of genes that provide the cellular response to the hypoxic environment 4.Hypoxia (low oxygen tensions) occurs when the normal oxygen supply to a tissue is disturbed. Environmental oxygen is both a nutrient and a signaling molecule, providing important cues for many cell types. Changes in environmental oxygen are sensed by a group of dioxygenases that control the activity of an essential transcription factor family known as the Hypoxia Inducible Factors (HIFs). The HIFs are composed of two subunits, α and β. There are three known isoforms of HIF-α  and multiple splice variants of HIF-1β. HIF-1β is constitutively expressed and not regulated by environmental oxygen levels5. However, hypoxia often exists as a gradient, and cells in a hypoxic environment are not subject to a uniform hypoxic stimulus. We describe an implementation of the Click-iT RNA imaging kits in an oxygen controlled workstation to examine global RNA synthesis at the single cell level in hypoxia.Current techniques for RNA analysis focus on the quantification of averaged values across a given cell population. Cells respond to a hypoxic stimulus by initiating the transcription of a myriad of genes that allow them to adapt to their hostile environment6.  Briefly, cells are treated with hypoxia and cultured in the presence of EU. They are then fixed and permeabilized and EU incorporation into nascent RNA is detected by chemoselective ligation of EU with an azide containing dye. A typical workflow for this reaction is shown in Figure 1B. We used the RNA imaging kit to examine RNA synthesis at the single cell level that resulted from treatment with hypoxia.The RNA imaging kit uses an alkyne-modified nucleoside, 5-ethynyl uridine (EU) and chemoselective ligation to enable detection of global RNA synthesis temporally and spatially in cells and tissues7-10. All of the reaction components are bioinert and the reaction requires no extreme temperatures or solvents. The click reaction negates the requirement for conventional radiolabelling and allows direct visualization of the results since the output is light. In addition, the detection molecule can easily penetrate complex samples allowing for multiplex analysis including antibodies for the detection of RNA-interactive proteins. This RNA imaging assay is compatible with organic dyes including Alexa Fluor and fluorescein (FITC).The small size of the alkyne tag enables efficient incorporation of the modified nucleoside into RNA specifically. The chemoselective ligation or 'click' reaction is highly efficient, fast and specifichttp://openmicroscopy.org/ . This microscope image visualization and analysis software enables access to, and use of a wide range of biological data, including the management of multidimensional, heterogeneous datasets. The client application allows remote visualization and analysis of complex biological image data11; we used it to quantify the visual changes in global and single cell RNA synthesis. These data and the steps required to analyze our RNA imaging experiment using this microscope image visualization and analysis software are shown below.We measured the change in RNA synthesis following treatment of our cells using an implementation of the open microscopy environment for remote objects (OMERO). OMERO is open-source software, available at 12. Investigating shorter hypoxia exposure times is therefore warranted and could detract from more complicated responses such as cell cycle and apoptosis.We looked at changes in global RNA synthesis following the treatment of human osteosarcoma (U2OS) cells with hypoxia for up to 24 hr. In all conditions, we detected cell-to-cell variation in the level of RNA production. Short times of hypoxia exposure did not result in significant changes to the level of nascent RNA in cells. However, exposure to 24 hr of hypoxia resulted in a significant increase in the amount of RNA produced in cells. Most of the cellular responses to hypoxia are measured following prolonged periods of exposure, such as 4 to 24 hr. However, some mechanisms are put in place much earlier, for example; NF-κB activation occurs within 5-15 min of hypoxia exposureTable 1 to culture human osteosarcoma (U2OS) cells. Prepare all reagents and conduct all tissue culture techniques in the laminar air flow hood to ensure sterilityGather the reagents and equipment listed in Prepare the culture medium as follows: Add 50 ml FCS, 5 ml L-glutamine and 5 ml penicillin/streptomycin to the DMEM to achieve final concentrations of 10% (FCS), 2 mM (L-glutamine), 50 U/ml (penicillin) and 50 U/ml (streptomycin). Warm the culture medium at 37 °C in a water bath.Sterilize several coverslips in 70% ethanol, allow to air dry and place one on the bottom of six 3.5 cm plates.Immerse the coverslips in 2 ml warmed complete DMEM (37 °C), pressing down with the coverslip forceps to ensure that they remain adhered to the bottom of the wells.Detach the U2OS cells from their tissue culture plate by washing once with 3 ml PBS, applying 4 ml warmed 0.05% trypsin-EDTA (1%) and incubating at 37 °C for 7 min.Resuspend cells in 6ml warmed complete DMEM to inactivate the trypsin-EDTA and count using a hemocytometer.5 cells to each of the 3.5 cm plates prepared in point 1.3.1. Gently rock the plate to ensure an even cell distribution. Allow the cells to adhere overnight prior to treatment.Plate 2 x 10Prepare the cells for treatment: (in the hypoxia chamber for hypoxia treated cells) 1 hr before the hypoxia treatment ends.Treat the cells by placing four 3.5 cm plates into the hypoxia workstation and leave the other two 3.5 cm plates in the incubator at 37 °C in normal oxygen conditions. Be ready to commence the RNA labelling assay under treated conditions Expose cells to hypoxia for 1 hr 15 min, 1 hr 30 min, 2 hr, and 24 hr. These time points are flexible and user-determined.Use a normoxic 3.5 cm plate as the positive control and a normoxic 3.5 cm plate treated with Actinomycin D  for 4 hr as the negative control. Actinomycin D is an inhibitor of global RNA synthesis.Hoechst 33342 is a known mutagen. DMSO is known to facilitate the entry of organic molecules into tissues. NaOH is corrosive. HCl is corrosive. Paraformaldehyde is highly toxic to all animals and can cause death in humans. Triton X-100 can cause skin irritation following direct contact. Actinomycin D is toxic when in contact with skin or swallowed. Take appropriate precautions when handling all hazardous chemicals.2O), dimethyl sulfoxide (DMSO), 10 M sodium hydroxide (NaOH), and 37% hydrochloric acid (HCl); Optional - pH indicator strips.Gather the following reagents that are required in addition to the Click-iT assay kit: phosphate buffered saline (PBS) pH 7.2 - 7.6, 3.7% paraformaldehyde (PFA) in PBS, 1% Triton X-100 in PBS, deionized water  and mix by pipetting or vortexing. Store both at -20 °C for up to one year.2O to component E to create a 10X solution of the RNA imaging kit reaction buffer additive. Store at -20 °C for up to one year, discard when solution develops a brown color.Add 2 ml dHPrepare the stock solutions of the RNA imaging kit as follows: Perform the remainder of this step and step 2.5 in the hypoxia chamber for cells treated with hypoxia. Dilute to 1X by adding an equal volume of this 2X working solution to the media containing cells. Incubate for 1 hr under treatment cell culture conditions.Labeling of Cells with EU: Prepare a 2X working solution of EU from the 100 mM stock prepared in step 2.3 in prewarmed complete medium (37 °C). The samples treated with hypoxia can be removed from the hypoxia chamber at this point. Remove the fixative and wash each well once with PBS (Take care to dispose of PFA waste responsibly). Perform the next steps at room temperature.Cell Fixation: Wash each well once with PBS and add 1 ml of the 3.7% PFA stock prepared in step 2.2 above under treatment conditions. Incubate for 15 min under treatment conditions. Permeabilization: Remove the wash solution and add 1 ml of 1% Triton X-100 in PBS to each well. Incubate for 15 min at room temperature. Remove the permeabilization buffer and wash each well once with PBS.2O.Prepare a fresh 1X working solution of RNA imaging kit reaction buffer additive by diluting the 10X solution (prepared in step 2.3) 1:10 in dHTable 2 for use immediately after preparation.Prepare the RNA imaging kit reaction cocktail according to protect from light.Remove the wash solution and add 500 µl of RNA imaging kit reaction cocktail to each sample. Incubate for 30 min at room temperature, Remove the RNA imaging kit reaction cocktail and wash once with 1 ml of RNA imaging kit reaction rinse buffer (component F), then remove the RNA imaging kit reaction rinse buffer.Labeled RNA Detection: Optional multiplex reaction: Perform additional antibody labeling of samples at this point according to manufacturer's recommendations.protect from light. Remove the Hoechst 33342 solution and wash the cells twice with PBS. Remove the wash solution and proceed to light microscopy.DNA Staining: Wash samples with PBS then remove the wash solution. Dilute the Hoechst 33342 (Component G) 1:1,000 in PBS. Add 1 ml of diluted Hoechst 33342 to each well and incubate for 15 min at room temperature, Place 10 µl of mounting medium on the center of a standard microscope slide.Place the coverslip with cell-side down on the center of the microscope slide to cover the aliquot of mounting medium. Take care to avoid the generation of bubbles in the mounting medium as this will obscure subsequent image acquisition.Protect from light. Samples may be stored at -20 °C following mounting.Stick the coverslip to the microscope slide by applying clear nail varnish to its perimeter. Allow to dry for 5 min at room temperature. Mount Coverslips: Table 3.Image acquisition: Acquire images using a wide-field microscope capable of fluorescence detection. Approximate fluorescence/excitation emission maxima for the Alexa Fluor dye and Hoechst 33342 bound to DNA are shown in Perform Imaging using a 40X/1.30 NA oil immersion lens and capture images with a cooled CCD camera.Deconvolve images using image processing software  prior to importing to the OMERO client. Normalize rendering settings for all images in the same experiment in the microscope image visualization and analysis software by applying the rendering settings from control normoxic cells to all other conditions.Use the Region Of Interest (ROI) tool in the microscope image visualization and analysis software  to select nuclei from each image. Obtain mean intensities for each ROI and calculate the average and standard deviation for each condition by choosing the intensity analysis option, including a number between 30-50 cells.Construct Scatter plots using a data graphing software  by plotting all individual mean intensity values obtained in step 4.2 to reflect variability between cells under each condition. Alternatively construct bar graphs to represent average mean intensity plus standard deviation of nascent RNA.Figure 1B. We used this reaction to quantitatively determine total RNA synthesis in U2OS cells following treatment with hypoxia on both an individual (single cell) and global level (population of cells). The cells were treated with hypoxia and cultured in the presence of EU. They were then fixed and permeabilized and EU incorporation was detected by chemoselective ligation of EU with an azide containing dye. This 'click' reaction results in a fluorescence emission that can be detected and quantified using fluorescence light microscopy. Figure 2A shows a typical result from this experiment. Fluorescently labeled cells are pseudocolored red and the image has been opened in the open microscopy environment for remote objects (OMERO). This microscope image visualization and analysis software has a built-in ROI tool that can be used to select individual sub-cellular structures and quantitatively analyze image intensity within the chosen region. A typical example of the data output following the use of this tool in the client program can be seen in Figure 2B; individual cell intensities are shown alongside a representative image from the experiment that serves as a visual check to ensure data validity.A schematic of the RNA imaging kit reaction is shown in Figure 2C. Plotting intensity data in this way is useful because it allows a rapid comparison of treatment results and clearly demonstrates the significant heterogeneity within a treatment cell population that is otherwise obscured using standard data representation. The response heterogeneity is interesting in this experiment because although a uniform hypoxic stimulus was applied to the cells, it is evident that every cell from the same monolayer population responds differently to this stimulus.The individual cell fluorescence intensities can be plotted as a scatter chart to show the distribution of the individual cellular intensities within the treated cell population. An example of this type of graph is seen in Figure 2D depicts the overall result from our experiment. Fluorescence intensities from all cells within a treatment population were averaged and then plotted in this bar chart. A student's t-test has been used to calculate the statistical probability of each treatment group differing significantly from the control. Interestingly, these data show that there is a statistically significant increase in global RNA synthesis over time when U2OS cells are treated with hypoxia . These data show that following longer periods of hypoxia treatment, the cell focusses its efforts on the production of RNA even in this hostile hypoxic environment. Actinomycin D treatment completely ablates the fluorescence intensity as expected.Figure 1. A. The HIF system responds to changes in cellular oxygen. In this simplified schematic hypoxia stabilizes the HIF heterodimer by preventing HIF-α hydroxylation (-OH) by the prolyl hydroxylase enzymes (PHDs) and its subsequent polyubquitination by von-Hippel Lindau protein (vHL). HIF stabilization results in the activation of myriad genes that help the cell respond to cellular hypoxia. B. RNA synthesis can be detected using the Click-IT assay; cells are labelled with EU after treatment the Click reaction detects RNA synthesis which can be quantified by fluorescence intensity and light microscopy. Please click here to view a larger version of this figure.Figure 2. A. Screenshot from OMERO to show typical microscopic output following RNA labelling. This microscope image visualization and analysis software can quickly extract intensity data on an individual cell level using its inbuilt ROI macro. A screenshot of the output from this command is shown in B. Global RNA synthesis can be inferred from individual cell intensity, the range of which can be seen in scatter plot C. A formal representation of these aggregate data can be seen D. *p < 0.01; **p < 0.05 and ***p < 0.001 and n > 30. Click here to view larger image.Table 1. Reagents and equipment required for culturing of U2OS cells.Table 2. RNA imaging kit reaction cocktails: Reference table detailing ratio of master mix components for RNA imaging kit reaction according to the number of coverslips to be stained.Table 3. Fluorescence/excitation emission maxima: Approximate fluorescence/excitation emission maximal for Alexa Fluor dye and Hoechst 33342 bound to DNA.We have described our use of an RNA imaging kit to examine the effects of hypoxia on RNA synthesis in osteosarcoma (U2OS) cells. This technique is relatively straightforward and provides data about global transcription at a single cell level. We modified the conventional protocol for this kit to incorporate labeling in a hypoxia chamber. To our knowledge this is the first report of the use of this technique to measure changes in RNA synthesis in hypoxia. The RNA imaging kit we used is suitable for experimentation in hypoxia since it requires no radioactive isotopes and is technically compatible with the limitations of working in a controlled atmosphere workstation. Common problems with this technique concern efficient fixation and labeling of the sample. It is extremely important that the PFA used to fix the sample is fresh and the wash steps that follow the 'click' reaction are performed as described to ensure low background staining of the sample. If background fluorescence becomes a problem it is recommended to wash once more with 1 ml RNA imaging kit reaction rinse buffer after step 2.7.4.The RNA imaging kit mentioned here represents a significant advance in RNA imaging technology in terms of ease of use and specificity and speed of reaction. This technique is primarily limited by the time it takes to label the sample. The RNA imaging kit requires a 1 hr labeling period that prevents the examination of rapid changes in global RNA that would usually occur within this timeframe. It is for this reason our first time point is restricted to 1 hr and 15 min. The technique is associated with few other limitations that largely relate to reagent compatibility with other fluorescent markers, for example, this RNA imaging kit is not compatible with phalloidin staining.6. The next advance in the field exploited means of immunochemistry to eliminate the necessity of radioactivity. RNA was labeled by incorporation of BrU followed by immunochemistry detection. However, efficient antibody binding required nucleic acid denaturation to improve access to DNA or RNA that caused loss of cell morphology and damaged the epitopes of many proteins, preventing their further detection with fluorescently labeled antibodies13. The introduction of 'click chemistry' technology simplified the detection step and the procedure compared to autoradiography and immunochemistry. The technology is based on azide-alkyne Huisgen cycloaddition reaction where terminal alkyne of 5-ethyniluridine binds with azide conjugated with the fluorophore in presence of Cu(I). The reaction is specific, rapid, does not require any additional steps, and is easily compatible with immunochemical detection of other cell constituents.All existing approaches of studying RNA synthesis in cells are based on incorporation of modified nucleosides into the nascent RNA and the detection of incorporated labels by different means. The pioneering approach was based on using radioactively labeled RNA precursors followed by detection with autoradiography. This method led to a discovery of cell cycle stages and other important findings; however, it has a lot of disadvantages such as the cumbersome nature of radioactivity work, long exposure times and low resolution images of autoradiographyUsing this RNA imaging technology we showed that cells, in the same monolayer population, have different RNA synthesis levels in response to hypoxia. We restricted our experiment to examine the effect of RNA production only; however, it is wholly possible with this RNA imaging kit to perform modified, additional multiplex reactions that allow for a more complex analysis of RNA production. For example, secondary staining using an antibody specific for markers of active transcription such as levels of phosphorylated RNA polymerase II or even components of the translation machinery to give an indication of the amount of this newly produced RNA that is converted into protein are possible. Additional proteins, such as Actin, a protein that should not change significantly with hypoxia, could be tested as an additional control. Furthermore, different cell types could be tested, as it is very likely that different cells will have different RNA synthesis rates and even different responses to hypoxia.Use of this RNA imaging kit is quite straightforward provided the steps are performed as described. Critical steps in the protocol involve cell plating, cell fixation and staining and image acquisition. It is important to plate the cells at the described density to prevent over or under confluence at experimentation which will significantly affect the result. It is also important to use fresh fixative, ensuring the best 'snapshot' of the cell is taken and to take care when washing the cells after staining to reduce the background to a level that is acceptable for efficient analysis. Finally, the method of image acquisition is vitally important to achieve images that are of a good enough quality for subsequent analysis. We recommend using the best light microscope available to examine samples optically such as the microscope used in this protocol which is available at most research intensive centers globally.J.R.S. is Founder of Glencoe Software, Inc., an open-source US-based commercial company that contributes to OMERO.


Lobaria pulmonaria as a model species, the objective of this study was to compare the effect of different rehydration conditions on the quantity and quality of RNA from the rehydrated thallus.The poikilohydric nature of lichens enables them to survive repeated episodes of desiccation by utilizing water when it becomes available. During rehydration, RNA-degrading endonucleases may be released, reducing RNA quantity and quality. Re-generation of a steady-state condition where RNA quantity and quality no longer fluctuate establishes a framework for development of new hypotheses for future investigations. Using Spectrophotometric measurements of total RNA and cDNA were performed for samples prepared from dry lichen or lichen after rehydration , with limited light and dark conditions, and at three temperatures  for some of these conditions. The results showed that rehydration of the thallus for 4 h at 20°C in light yielded the highest concentration and quality of RNA. A higher RNA concentration was obtained in light than in dark conditions, but the RNA quality was unaffected.This study suggests that allowance of 4 h for thallus rehydration should be adequate to ensure complete recovery of transcription. After 4 h at 20°C further studies can be carried out on the RNA in this model species. Water scarcity limits survival for most vascular plants because they cannot tolerate extensive dehydration. Poikilohydric organisms such as lichens and mosses have no mechanism to prevent dehydration, but can recover from low cell water content without physiological damage. Recovery of metabolic activity, such as transcription, upon rehydration has been studied in the vascular resurrection plant and moss , and is Lobaria pulmonaria is a foliose (leaf-like) lichen found in old growth forests  na lacera. The higR. lacera and in aL. pulmonaria thalli 4 h after rehydration, as determined by direct assessment of total RNA as well as by examination of cDNA quality. Such knowledge simplifies sample collection and in situ processing, allowing much greater flexibility for storage and transport of specimens without use of specialized reagents to assure RNA integrity. Experimentation to further our understanding of this model organism and other poikilohydric lichens may be pursued with some confidence that steady state conditions can be reached.A steady-state condition that may be representative of the natural lichen is attained by nifH: Nitrogenase gene; mtr SSU: Mitochondrial small subunit gene; Lpu_27: A simple sequence repeat locus; A260/280: Ratio of absorbance at 260 nm and 280 nm.cDNA: Complementary DNA; DW: Dry weight; The authors declare they have no competing interests.JD contributed to study design, carried out all lab work and drafted the manuscript. VM collected the specimens, provided critical advice on the analysis and interpretation of data, and made significant revisions to the manuscript. MN provided funding, designed the study, provided advice and training to JD on the molecular biology, and made critical revisions of the manuscript. All authors have given final approval of the manuscript.


Although studies of self-rated health (SRH) are conducted widely in developed countries, comprehensive assessments of the determinants of SRH in Chinese are scarce, particularly for working Chinese individuals. Determinants of SRH might differ among worksites based on differences in the nature and stress associated with different jobs, work intensity, and the lifestyles of employees.Two thousand and forty questionnaires that addressed SRH, demographic characteristics, lifestyle, and the psychosocial work environment were administered to employees at two worksites. A total of 1644 subjects provided complete data for analysis (80.6% response rate).Participants from government departments had significantly better SRH than did those from high-tech enterprises . Lifestyles were significantly less healthy at government departments compared with high-tech enterprises, whereas the psychosocial work environment was better. The results of unadjusted and adjusted models revealed differences between the potential health-influencing factors of participants based on their type of worksite. In logistic regression models, gender was strongly associated with SRH in all participants, whereas length of service was correlated with SRH only in participants from high-tech enterprises. In high-tech enterprises, good SRH was less common in physically inactive subjects vs. physically active participants (OR = 0.561). In government departments, passive smoking was negatively associated with SRH significantly. Social capital (OR = 1.073) and job control (OR = 1.550) were positively correlated with SRH in high-tech enterprises. Job control was the only psychosocial factor significantly associated with SRH in government departments.Participants from different types of worksite reported different SRH, healthy lifestyles, and psychosocial work environments. Moreover, the association between SRH and demographic characteristics, lifestyle, and the psychosocial work environment significantly differed by type of worksite. SRH is generally assessed by a single item index or a single survey question inviting respondents to provide a subjective assessment of their health using some form of a five-point scale . In a wiThe prevalence of chronic diseases such as cardiovascular disease and diabetes has been increasing rapidly in recent years along with recent economic development , suggestCompared with studies of Chinese populations, those conducted in developed countries indicated more valuable and in-depth results. Some studies on the determinants of SRH revealed that positive health-related lifestyles might have a positive effect , and thaIn this study, we assessed participants from two representative types of worksite, including 10 government departments and two high-tech enterprises. These types of worksite have comparable features such as a relatively stable time arrangement, mental labor, and high education requirement. However, high-tech enterprises were perceived as high job demand and fast work rhythm because employees had to update their specialized knowledge to accomplish sophisticated duties on time without error. In contrast, civil servants are considered to have the most stable job in China. Most civil servants are responsible for drawing up plans and providing guidance and supervision for relevant activities. Although civil servants are perceived to have an easy job, they have inflexible schedules and a lack of decision-making ability.The aim of this study was to investigate and compare the association between SRH and demographic characteristics , lifestyle , and psychosocial work environment  between participants from two representative types of Chinese worksite.Written informed consent statement forms were obtained from respondents, and the right to withdraw and autonomy of the responses were explained. The study received ethical approval from the ethics committee of School of Public Health of Fudan University, China.Twelve interviewers underwent a training lesson in which the standard guidelines for the investigation were emphasized. Each interviewer was responsible for collecting the self-administered questionnaires from each worksite during the working day from July to September 2012.In this study, we assessed participants at two representative types of worksite. The participants consisted of 2040 employees who worked in Shanghai. One thousand, six hundred and forty-four subjects provided complete data for analysis, which was a response rate of 80.6%. Participants were recruited from 12 different worksites: 10 government departments and two high-tech enterprises.Self-reported demographic variables included gender, age, education, length of service, and marital status. Age and length of service were separated into four and five categories, respectively. The four age categories were <30, 30 - 39, 40 - 49, and >50 years. The five categories for length of service were <5, 5 - 9, 10 - 14, 15 - 19, and ≥20 years.SRH was the main health outcome of this study. Respondents were asked to rate their own general health on a five-point scale ranging from perfect to poor. As the dependent variable in logistic regression, the original variable was dichotomized according to the distribution of SRH, with 1 representing perfect, very good, and good health, and 0 representing fair and poor health.Four health behaviors and two potential influencing factors were included in the study. Current smoking status , passive smoking (yes/no), alcohol intake , and physical activity  were assessed using self-reporting questionnaires. People who had smoked more than 100 cigarettes were defined as smokers. Passive smokers were respondents who had been exposed to others’ smoke for more than 15 minutes in the last week. The respondents who had drunk alcohol were defined as alcohol drinkers. The self-reported data for physical activity were collected from the Chinese version short IPAQ , which was acceptably reliable (ICC of 0.79 and %CV of 26%) .The IPAQ short form asked about three specific types of physical activity (PA) including walking, moderate-intensity activities such as dancing, cycling, and performing tai chi, and vigorous-intensity activities such as swimming and playing basketball. The following values were used to analyze IPAQ data: walking = 3.3 metabolism equivalents . Calculating of the total score for the short form required summation of the duration (in minutes) and frequency (days) of walking, moderate-intensity activities, and vigorous-intensity activities. Three levels of physical activity were proposed to classify the populations: low, moderate, and high. The two criteria for classification as “high” were (a) vigorous-intensity activity on at least 3 days achieving a minimum total physical activity of 1500 MET-minutes per week, or (b) 7 or more days of any combination of walking, moderate-intensity, or vigorous-intensity activities achieving a minimum total physical activity of 3000 MET-minutes per week. The pattern of activity classified as “moderate” was either of the following criteria: (a) 3 or more days of vigorous-intensity activity of at least 20 minutes per day, (b) 5 or more days of moderate-intensity activity and/or walking for at least 30 minutes per day, or (c) 5 or more days of any combination of walking, moderate-intensity, or vigorous intensity activities achieving a minimum total physical activity of 600 MET-minutes per week. Individuals who did not meet the criteria for “moderate” or “high” were considered to have a “low” level of physical activity.The social capital scale measured both the cognitive and structural components of social capital using eight items . The intParticipants were classified into two groups according to the type of worksite: high-tech enterprise employees and government department employees. Descriptive analysis, analysis of variance, and chi-squared tests were conducted to compare the differences between SRH and potential health-influencing factors between the two groups.Multiple logistic regression analysis was conducted to examine the factors that influenced SRH among participants from different types of worksite. In either group, a binomial logistic regression analysis was performed in three steps following the theoretical framework presented in the Introduction. First, background variables  were included in models A and B. Next, lifestyle factors  were added to models C and D. Finally, psychosocial work environment factors  were introduced into models E and F. Unadjusted and multivariate-adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated from logistic regressions to examine the factors that influence SRH. The 2log-liklihood and significance of the Hosmer-Lemeshow test were also reported on each model to allow us to evaluate the adequacy of changes in the model and additional variables. For all analyses, statistical significance was set at 0.05. Statistical analysis was performed using the Statistical Package for Social Sciences (IBM SPSS 20).2 = 22.125, P < 0.001). The distribution of age differed by type of work. In high-tech enterprises, nearly half of the participants were <30 years of age, compared with only 17.2% in the government departments . The proportions of all other age groups were similar. The distribution of length of service had similar characteristics as age . A total of 62.5% of participants from high-tech enterprises had been married, compared with 90.3% of government workers. More than half of the respondents had a bachelor’s or higher degree in both groups.Data were collected from 2,040 participants from 12 different worksites comprising 10 government departments and two high-tech enterprises. The demographics of the study sample are reported in Table P = 0.001). The proportion of individuals who reported good SRH decreased significantly with age . Similar characteristics were found among the different groups of length of service . The proportion of married participants who reported good SRH was lower than that of the unmarried participants . The proportion of individuals with a bachelor’s degree who reported good SRH was higher than the other groups .Table Table For potential health influencing factors, participants from government departments had significantly more social capital, lower job demand, and more professional support than did those from high-tech enterprises. The scores for job control were comparable. Compared with participants from high-tech enterprises, respondents from government departments led significantly unhealthier lifestyles with less physical activity, increased smoking and passive smoking rates, and more alcohol consumption.2 = 20.261, P < 0.001). A total of 68.7% of participants who drank alcohol monthly reported good SRH, which was the highest among the three groups . The proportion of the passive smokers who reported good SRH was lower than that of non-passive smokers . There were also significant differences between SRH in groups of all the psychosocial work environment factors, except for job demand.Table Logistic regression analyses were used to identify the potential factors that influenced SRH Tables  and 5. HGender had a strong association with SRH in all participants, whereas length of service was associated with SRH in participants from high-tech enterprises. Participants employed for <5 years were more likely to have good SRH (OR = 3.113) compared with those employed for >20 years. Age and length of service were also associated with SRH in different directions. Moreover, these associations varied according to type of worksite. For participants from high-tech enterprises, people <50 years old were less likely to have good SRH (OR <0.5) than subjects >50. Consistent with this, participants who were employed for <20 years were more likely to have good SRH (OR <0.5) compared with those employed for >20 years. For participants from government departments, people <50 years old were more likely to have good SRH (OR >1.0) than respondents >50. However, individuals who were employed for <20 years were less likely to have good SRH (OR <1.0) than those employed >20 years. Although there was some variation between different subgroups of age and length of service, there were no significant differences.Some lifestyle factors were also related to SRH after adjusting for demographic variables. For participants from high-tech enterprises, good SRH was less common (OR = 0.561) among physical inactive participants compared with highly physically high active ones. For participants from government departments, passive smoking was negatively correlated with SRH significantly. The effects of lifestyle factors were mainly independent, and did not change when psychosocial factors were introduced into the model. Adding the lifestyle factors into the model attenuated the association between length of service and SRH in participants from high-tech enterprises. However, for participants from government departments the association between gender and SRH was strengthened after the addition of lifestyle factors.Psychosocial factors were introduced into the final models. Some psychosocial factors were associated with SRH after adjusting for demographic variables and lifestyle factors. For model E, social capital (OR = 1.073) and job control (OR = 1.550) were positively associated with SRH in participants from high-tech enterprises. For model F, job control was the only psychosocial factor significantly associated with SRH in participants from government departments. After considering psychosocial factors, the association between education and SRH was strengthened in participants from high-tech enterprises, whereas the association was attenuated in participants from government departments.We found a modest level of good SRH among all participants (64.6%), which varied significantly by type of worksite. Lim et al. studied 6236 individuals aged ≥18 in Singapore (an urban area) and found a level of good or excellent SRH in 77% of respondents . SimilarCompared with civil servants, participants from high-tech enterprises led a significantly healthier lifestyle. Specifically, they were more physically active (69.6 vs. 61.3%), smoked less 19.9 vs. 31.4%), had less alcohol intake (37.6 vs. 51.3%), and less passive smoking (37.6 vs. 71.3%). Another study showed that civil servants were more physically active than company staff (67.7 vs. 67.1%) . These i.4%, had We found that civil servants reported significantly better psychosocial work environments, including more social capital, lower job demand, and more support at work than participants from high-tech enterprises. However, the scores of job control were comparable. A study of Chinese civil servants reported that nearly half of respondents perceived high job stress . HoweverBased on these results, civil servants had worse lifestyles than participants from high-tech enterprises. Moreover, the proportion of male participants was higher in high-tech enterprises than in government departments. The proportion of young participants from high-tech enterprises was also higher. Several studies in Shanghai showed that males had a worse lifestyle than females, whereas young people led a worse lifestyle than middle-aged individuals , 39. TheIn addition, civil servants reported better a psychosocial worksite environment than participants from high-tech enterprises. The difference in the psychosocial worksite environment between government departments and high-tech enterprises was easy to understand. High-tech enterprises were perceived to have a high job demand and fast work rhythm because employees are required to update their specialized knowledge and accomplish sophisticated duties on time without error. In contrast civil servants, who are responsible for drawing up plans and providing guidance and supervision for relevant activities, are considered to have an easy job. In the current analyses civil servants had a better SRH than high-tech enterprise employees. It is possible that the psychosocial worksite environment might be more important for determining SRH than health behaviors. Although it remains unclear what is behind this interesting phenomenon, two characteristics might play a role. First, in our opinion, SRH provides a subjective assessment of one’s health, which is different from an objective health laboratory examination. In other words, subjective factors might influence an individual SRH more than objective factors. In the current study, health behaviors and psychosocial worksite environment were both measured by self-reported questionnaires rather than objective methods. However, health behaviors including physical activity, smoking, passive smoking, and alcohol drinking were reported based on participants’ objective behaviors rather than personal feelings. Personal feelings might have more effect on the measurement of a psychosocial worksite environment than health behavior. Second, determining the outcome of healthy or unhealthy behaviors will take a long period of time. For example, smokers will not realize the harmful effect of smoking until they suffer from adverse symptoms. However, the psychosocial worksite environment such as job demand and job control will affect people’s moods and feelings immediately.Our study indicated that gender had a strong association with SRH among all participants, whereas length of service was associated with SRH in participants from high-tech enterprises when only demographic factors were included in the models.Consistent with our findings, other studies reported that males were more likely to report good SRH than females , 15, andWhen lifestyle factors were introduced into the models, different factors were related to SRH after adjusting for demographic variables in the two types of worksite. Among high-tech enterprise employees, physical activity was positively correlated with good SRH significantly. However, passive smoking was negatively associated with good SRH in civil servants. Some studies also found a similar association between physical activity and SRH , 41. In Psychosocial factors were introduced into the final models. The determinants of SRH differed by type of worksite. For employees from high-tech enterprises, SRH was related to gender, education, physical activity, social capital, and job control. However, in workers from government departments SRH was associated with gender, age, passive smoking, and job control. Among the psychosocial factors, job control was positively associated with SRH in participants from both type of worksite. Consistent with our findings, other studies reported that low job control was associated with poor SRH in various working people including nurses and municipal employees , 42; howIn general, our results revealed some differences between the potential health-influencing factors of participants according to type of worksite in both unadjusted and adjusted models. There were different main influencing factors of health between the two work groups. For employees from high-tech enterprises, a lack of social capital and professional support should be taken seriously. If feasible, lowering employees’ job demands might be an effective way of improving their health. However, for government departments the priority should be to help civil servants establish healthy lifestyle habits, including enough physical activity and cessation of smoking. In addition to health education, environmental support, health culture, and leaders’ health behaviors play a critical role in improving civil servants’ health.There are some limitations to our study. First, the direction of causality could not be addressed due to the cross-sectional study design. Second, all measures were based on self-reports, even though the measures have been validated , 26, 44.Participants from different types of worksite reported different SRH, healthy lifestyles, and psychosocial work environment. The association between SRH and demographic characteristics, lifestyle, and psychosocial work environment differed by type of worksite significantly. Among participants from high-tech enterprises, gender, education, length of service, physical activity, social capital, and job control were associated with SRH. Among participants from government departments, gender, age, passive smoking, and job control were associated with SRH. The psychosocial work environment might be more important for determining better SRH than health behaviors among employees. It is necessary to develop interventions that focus on different areas to improve employees’ health according to the type of worksite and characteristics of the employees.