RNA bacteriophages like Qbeta and MS2 are well known for their high mutation rate, short infection cycle and strong selection against foreign inserts. The hammerhead ribozyme (HHRz) is a small self-cleaving RNA molecule whose active residues have previously been identified by mutational analysis of each individual base. Here the functionally important bases of HHRz were determined in a single screening experiment by inserting the HHRz into the genome of MS2.satellite Tobacco ringspot virus was cloned into the genome of RNA bacteriophage MS2. Sequence analysis of the surviving phages revealed that the majority had acquired single base-substitutions that apparently inactivated the HHRz. The positions of these substitutions exactly matched that of the previously determined core residues of the HHRz.The minimal HHRz of Natural selection against a ribozyme in the genome of MS2 can be used to quickly identify nucleotides required for self-cleavage. These pdeletion . Certaindeletion ,5. So, Isatellite Tobacco ringspot virus HHRz and an inactive control were cloned into MS2 cDNA , one of the best-studied ribozymes, was originally discovered in plant virus satellite RNA  and viroA Figure B,C. ActiE. coli to yield separate plaques. Sequence analysis of individual plaques from the inactive HHRz containing phage revealed no sequence changes (13 out of 13 samples). Of 50 plaques taken from the active HHRz clone, 44 had acquired a single mutation, one had two mutations, and one had a single A-deletion  led to an amino acid change in the extension (Ser to Ala). These data are in agreement with an earlier study on C-terminal extensions of the A protein .The data show that natural selection against a ribozyme in the genome of MS2 can be used to quickly identify nucleotides that contribute to self-cleavage: the selected mutants give an accurate description of the previously determined core residues of the HHRz. Of course to obtain a complete overview of all four possible bases at each position, especially the less frequent base transversions, one needs to analyze substantially larger numbers of mutants.6 or 107 plaque forming units per ml would suffice to screen for mutants that may have lost this loop-loop interaction.The natural TRSV HHRz has a loop-loop interaction that increases catalytic activity over 100-fold -14. AlthThe present method can be used to investigate other ribozymes, provided that their size is smaller than about 100 nts, they possess a large degree of secondary structure - MS2 displays a strong selection against large single-stranded inserts  – and cl12 plaque forming units (pfu) per mL culture after overnight growth [pMS2000 contains a complete cDNA copy of the phage MS2 genome under transcriptional control of a thermoinducible promoter and produces 10GCTAGCGTAATACGACTCACTATAGGGTATCTTGAACCCACTAGG, MS2 sequence homologous to nts 964–983 in bold, NheI restriction site in italics) and MSLuc2 . The two PCR fragments were digested with NheI and BamHI and cloned into a Firefly luciferase reporter vector (details will be described elsewhere). After linearization of the resulting plasmids with ApaI and purification of the DNA, RNA was synthesized by T7 RNA polymerase transcription using the RiboMAX Large Scale RNA Production System (Promega). Products were assayed by electrophoresis on an Ethidium bromide containing agarose gel.DNA fragments covering the region 964–1421 of the MS2 genome were produced by PCR on the pMS2000 variants containing the active and inactive variant of the HHRz using primers MS2up3  which contains a kanamycin selectable F-factor, needed for phage attachment. Individual plaques were lanced with a yellow tip and transferred to cell culture tubes containing 1 mL of LB supplied with kanamycin and F+ cells (OD650 ~ 0.05) and grown overnight at 37˚C. Phage present in 1 mL of supernatant were precipitated with PEG and phage RNA was extracted as described [Phages were plated onto lawns of escribed . cDNA waThe author declares that he has no competing interests.RO did all the experiments and wrote the manuscript.


Five simulated skull models were created to simulate typical clinical conditions experienced while programming implanted adjustable ventriculoperitoneal hydrocephalus shunt valves. These models were created with physician feedback and evaluated by 13 health care professionals. Based on tactile evaluations and qualitative feedback, three models were selected as most clinically relevant. An optical measurement and valve setting method were developed to characterize programming tool movements during valve programming procedure and usability testing that evaluated over 50 health care professionals that program hydrocephalus valves.The skull models used a variety of synthetic tissues and thicknesses simulating a 3mm scalp thickness protruding valve model (such as younger or older patients with thin skin), a 7mm scalp thickness model (that characterizes ‘average’ patients) and a 10mm thick model . Different colors of ultraviolet fluorescent invisible ink markers were used to draw dot pairs relative to the valve center. Precision drilled tool kits (precision machined pairs of holes) were developed to characterize angular offset in addition to actual offset from valve center. Models were covered in plastic wrap enabling the same models to be reused throughout usability studies minimizing error. Various cameras were used (video and still) with visible and ultraviolet light photography throughout usability testing.The printed grid overlays were on average 0.05mm +/- 0.35 mm offset compared to the drawing; The machined toolset hole centers were on average -0.03mm +/- 0.40 mm offset compared to the drawing; The markers dots were on average 0.15mm +/- 0.30 mm offset compared to the drawing; The valve center was on average 0.43mm +/- 0.22mm offset from the printed grid overlay and had a difference in angle of -0.26 +/- 1.37 degrees from the actual position of the valve. Real time valve setting was enabled with an endoscope placed underneath the programmable valve.It is very important to accurately characterize human factors while developing medical devices. Clinically relevant models and these measurement methods enabled characterization of programmable hydrocephalus valve tool kit usability.


Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects. Viruses are highly abundant in most biological systems and are characterized by an extraordinary diversity . Large-sDengue virus and Chikungunya virus , in southeastern Brazil. Laboratory colonies of L. longipalpis sandflies were derived from wild-caught animals captured in the city of Teresina, in northeastern Brazil. Drosophila libraries were prepared from wild-type laboratory stocks that were infected with Vesicular stomatitis virus, Drosophila C virus or Sindbis virus as described previously  on a denaturing PAGE before being used for construction of libraries as previously described ( nt on a http://hannonlab.cshl.edu/fastx_toolkit/index.html). Small RNA reads with phred quality below 20, shorter than 15 nt after trimming of adaptors or containing ambiguous bases, were discarded. In the case of long RNA libraries, raw sequenced reads were submitted to quality filtering using fastx_quality_filter. Reads with phred quality below 20 or containing ambiguous bases were discarded. Remaining sequences from small or long RNA libraries were mapped to reference sequences from transposable elements, bacterial genomes (2739 complete genomes deposited in Genbank) and host genomes  using Bowtie (version 1.1.1) for small RNA libraries or Bowtie2 (version 2.2.4) for long RNA libraries  (Drosophila genome (version v5.44) was downloaded from flybase.org. The latest versions of mosquito (Liverpool strain L3) and sandfly (Jacobina strain J1) genomes were downloaded from VectorBase (https://www.vectorbase.org/). Sequences of transposable elements were obtained from TEfam (http://tefam.biochem.vt.edu/tefam/). Remaining sequenced reads that did not map to transposable elements, host or bacterial genomes, referred to as processed reads, were used for contig assembly and subsequent analysis.Raw sequenced reads from small RNA libraries were submitted to quality filtering and adaptor trimming using fastx_quality_filter and fastq_clipper respectively, both part of the fastx-toolkit package (version 0.0.14)  ,32. The http://bioinformatics.net.au/software.velvetoptimiser.shtml). For long RNA libraries, contig assembly was performed using a fixed k-mer value (k-mer 31) or an automatically defined k-mer value from 15 to 91. For each library, results from parallel contig assembly strategies were merged using CAP3 (version date 12-21-07) with max gap length in overlap of 2 and overlap length cutoff of 20 (Processed reads were utilized for contig assembly using Velvet (version 1.0.13) . Assemblff of 20 . Resultsff of 20 , requiriE-value smaller than 1e−5 for nucleotide comparison or 1e−3 for protein comparison were considered significant. Viral genomic segments were classified as described (Assembled contigs were characterized by sequence similarity (nucleotide and protein) to known sequences, with analysis of conserved domains if detected, and also examined for the presence of ORFs. We used BLAST for sequence similarity searches against non-redundant NCBI databases (nucleotides and protein). InterproScan (version 5.3–46.0)  and HMMeescribed .For pattern-based analysis, processed small RNA reads were mapped against contig or reference sequences using Bowtie allowing one mismatch. Small RNA size profile was calculated as the frequency of each small RNA read size from 15–35 nt mapped on the reference genome or contig sequence considering each polarity separately. We used a Z-score to normalize the small RNA size profile and to plot heatmaps for each contig or reference sequence using R (version 3.0.3) with gplots package (version 2.16.0). To evaluate the relationship between small RNA profiles from different contig or reference sequences, we computed the Pearson correlation  of the Z-score values. Similarities between small RNA profiles were defined using hierarchical clustering with UPGMA as the linkage criterion. Groups of sequences with more than one element with at least 0.8 of Pearson correlation between each other were assigned to clusters. The density of small RNA coverage was calculated as the number of times that small RNA reads covered each nucleotide on the reference genome or contig sequence. Small RNA size profile and density of coverage were calculated using in-house Perl (version 5.12.4) scripts using BioPerl (version 1.6.923) and plotted using R with ggplot2 (version 1.0.1).About 200 ng of total RNA extracted from insects was reverse transcribed into cDNA using MMLV reverse transcriptase. cDNA or DNA were subjected to polymerase chain reaction (PCR) reactions using specific primers. Oligonucleotide primers are listed in Supplementary Table S1 and were designed according to contig sequences obtained from our assembly pipeline. PCR products were subjected to direct Sanger sequencing.D. melanogaster and wild populations of A. aegypti mosquitoes and L. longipalpis sandflies, two important vectors for human pathogens (Supplementary Table S2). Drosophila libraries were prepared from laboratory strains infected with three distinct viruses, Drosophila C virus (DCV), Sindbis virus (SINV) and Vesicular stomatitis virus (VSV) in order to help optimize our virus detection pipeline for small RNA sequences. Small RNA libraries were prepared from whole insects with no sample manipulation prior to RNA extraction to minimize risks of sample contamination in the laboratory, which is essential when processing field samples. After sequencing of small RNA libraries, data were processed to enrich for potential viral sequences by removing sequences derived from host and bacterial genomes. Host sequences corresponded to the vast majority of small RNAs (73–92%) in our libraries but a substantial percentage of sequences (3.4–14% of libraries) remained after these processing steps . Thus, our approach could detect viruses known to be present in flies, although detection was limited by the number of viral small RNAs. We observed 1572 small RNAs derived from DCV that only allowed assembly of one contig covering 0.8% of the viral genome . In contrast, 53 620 and 9588 small RNAs derived from VSV and SINV, respectively, allowed assembly of multiple independent contigs that covered 81.1 and 23.4% of the respective genomes . Thus, high coverage of viral genomes is important to allow contig assembly from overlapping small RNAs.Libraries from infected D. melanogaster, A. aegypti and L. longipalpis small RNA libraries were utilized for sequence similarity searches against the NCBI non-redundant databases (nucleotide and protein). The vast majority of contigs assembled in all nine small RNA libraries (10 577 out of 11 806) did not show any significant sequence similarity and are hereafter referred to as unknown . The remaining 1229 non-redundant contigs were classified according to the taxon assigned to their most significant BLAST hit  showed significant similarity to viral sequences in reference databases Figure . These rAedes mosquitoes we detected contigs that belong to a novel strain of Phasi Charoen Like-virus (PCLV), a bunyavirus previously identified in mosquitoes from Thailand (Laem Singh virus (LSV) and two other recently described tick viruses, Ixodes scapularis associated virus 1 and 2  (Humaita-Tubiacanga virus (HTV) to reflect the origin of its host.In gure S3A). In addiure S3B) , all of Cypovirus from the family Reoviridae . These viruses were named Lutzomyia Piaui reovirus 1 (LPRV1) and Lutzomyia Piaui reovirus 2 (LPRV2) to reflect their host and geographical location. Analyses of the other viral contigs assembled from sandfly libraries that showed similarity to nodaviruses suggest they belong to a novel virus related to Nodamura virus and a member of the genus Alphanodavirus . This novel virus was named Lutzomyia Piaui nodavirus (LPNV).In sandflies, we observed several non-redundant contigs showing similarity to reoviruses and nodaviruses Table . Specifiae Table . This nuDrosophila laboratory stocks we used already carried unrecognized viral infections . This virus was named Drosophila reovirus (DRV). Another viral contig showed similarity to Acyrthosiphon pisum virus but could not be assigned to any known viral families by phylogeny . This virus was consequently named Drosophila uncharacterized virus (DUV).In fruit flies, we detected contigs that showed similarity to two viral families unrelated to the viruses used for experimental infections. That suggested the ns Table . One setDrosophila, Aedes or Lutzomyia. These results indicate that our strategy is not prone to generate artifacts.Sequences corresponding to all seven potential new viruses were successfully amplified by PCR from reverse transcribed RNA but not from DNA Figure . Sanger A. aegypti populations used to prepare small RNA libraries (Supplementary Table S2). This allowed us to directly compare results from large scale sequencing of small and long RNAs from the same samples without manipulation prior to RNA extraction. The number and length of sequences obtained with long RNA libraries resulted in 10.4-fold more data compared to small RNA libraries. As a result, long RNA libraries generated a total of 1 011 347 contigs with N50 of ∼136 nt compared to 6066 contigs with N50 of ∼48 nt for small RNA libraries  but phylogenetic analysis suggests the two viruses are considerably distinct . Phylogenetic analysis would seem to suggest that contig Aae.92 belongs to a viral family distinct from the two viruses that were also found in mosquitoes, PCLV and HTV. However, we note that DAV is an unusual virus, whose RdRP and capsid proteins show similarity to different viral families   of 1609 nt predicted to encode a protein with a coat domain (PF00729). This contig showed similarity with the capsid protein of gure S5) . Notablygure S5) . Hence, Lutzomyia (Llo), Drosophila (Dme) or Aedes (Aae). Small RNA size profile was determined for all 79 unknown contigs and compared to previously characterize viral contigs using hierarchical clustering. This analysis generated 17 clusters containing more than one element, which were numbered sequentially according to the position in the heatmap. We observed that 72 out of the 79 unique unknown contigs were grouped in 11 different clusters of similarity  Figure . Thus, wy Figure . InteresLutzomyia libraries were grouped in three separate clusters that showed clearly distinct small RNA patterns .Unknown contigs found in V Figure . ClusterV Figure . Since tAedes libraries, 24 out of 27 unknown contigs were grouped in six different clusters  that showed high correlation to each other and a small RNA size profile consistent with mosquito piRNAs (27–28 nt peak in the size profile)  Figure  20,48).,48.AedesThe pattern of small RNAs generated by the host response depends on virus characteristics such as genome structure, tissue tropism or strategy of replication. Thus, besides identifying viral contigs, the small RNA size profile may also provide specific information on the biology of each virus. For example, RNA viruses tend to have a very homogenous small RNA coverage of the viral genome while DNA viruses show clear hotspots of small RNAs ,22,23. AWe also noticed that HTV and PCLV showed very distinct small RNA size profiles despite being sometimes found in the same mosquitoes Figures  and 3A. Flock House virus (FHV). Indeed, the B2 protein encoded by FHV is a powerful suppressor of silencing that blocks the RNAi pathway , Mosquitoe X virus (MXV) and Cell fusion agent virus (CFAV) in Aag2 cells, MXV and Insect Iridescent virus- 6 (IIV6) in U4.4 cells and Mosquito nodavirus (MNV) in adult mosquitoes (Supplementary Table S3). Notably, a 1130 nt sequence corresponding to MNV was originally identified by another small RNA-based analysis pipeline in the library from adult mosquitoes . Contig AaeS.81 has one complete ORF of 397 aminoacids and a second incomplete ORF that contains a RdRP_3 conserved domain (PF00998), the same domain found in the MNV contig  ,25. Sequsquitoes . Using tsquitoes . This 19squitoes . In addig Figure . The smag Figure . These rg Figure . In agreIn these published libraries, our pipeline also assembled a total of 1673 unknown contigs. Small RNA profiles were analysed for eight unknown contigs longer than the N50 observed for viral contigs (208 nt). Regarding the small RNA size profile, most viral contigs identified in published insect datasets were grouped in a single large cluster showing a 21 nt peak size consistent with typical siRNAs Figure . The lacMegavirus terra 1 . High correlation of the small RNA size profile suggests U4.4.84 and U4.4.85 have the same origin. We also found small RNAs derived from contigs U4.4.84 and U4.4.85 in the small library prepared from Aag2 cells in the same laboratory as U4.4 cells . These observations could suggest an infectious virus that contaminated both cell cultures since small RNAs derived from contigs U4.4.84 and U4.4.85 were not observed in Aag2 cells from our own laboratory (data not shown). Another five unknown contigs were assembled in the library from Aag2 cells but showed small RNA profiles consistent with piRNAs suggesting these might represent repetitive regions absent from the mosquito genome.We also identified two unknown contigs of 390 and 363 nt in U4.4 cells, U4.4.84 and U4.4.85, that showed a size profile similar to several viruses grouped together Figure . Contig Arabidopsis thaliana leaves infected with Turnip mosaic virus (TuMV), grouper fish GP cells infected with Singapore grouper iridovirus (SGIV), mouse lungs infected with Severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse embryonic stem cells infected with Encephalomyocarditis virus (EMCV)  –58. Samps (EMCV)  and E. NMCV –58. y Figure . This isy Figure  16,59).,59.ArabiArabidopsis, viral contigs assembled in mouse and fish libraries were among the shortest . No. NoArabit Figure . The sizt Figure  and E. TWe also directly compared virus identification from different RNA fractions prepared from mouse lungs infected with SARS-CoV ,60. SARSWe also observed that very few contigs assembled by our pipeline in small RNA libraries from mice, fish and plants were unknown sequences Figure  and B. TIn this study we describe a powerful approach based on small RNAs that allows for successful identification of viruses without any prior information about their presence. The majority of the viruses we identified potentially represent new species, illustrating the power of our strategy. Importantly, our results strongly indicate that virus identification from small RNAs provides four notable advantages compared to other metagenomic strategies. Firstly, preparation of small RNA libraries requires little sample manipulation and no column filtration steps before RNA extraction. This minimizes the chance of sample contamination or bias that can affect virus discovery by metagenomic studies . SecondlA large part of our strategy was based on the diversity of virus-derived small RNA profiles observed in infected insects. Although virus-derived small RNAs profiles can be very heterogeneous in infected insects, only the production of 21 nt long virus-derived siRNAs has classically been considered a hallmark of antiviral immunity ,20–23,61Bunyaviridae and Reoviridae) that include several mammalian pathogens. Future studies should evaluate the presence of these viruses in wild mosquito and sandfly populations in Brazil as a potential threat for humans and livestock. In addition, these viruses could affect the ability of vector insects to carry other human pathogens such as Dengue virus and Leishmania, naturally transmitted by mosquitoes and sandflies, respectively. Together our results indicate that sequencing of small RNAs is a powerful virus surveillance strategy in research laboratories as well as natural settings.Using our small RNA-based approach we characterized the virome of laboratory stocks of fruit flies and wild populations of two vector insects, mosquitoes and sandflies. These included six novel viruses and a strain of PCLV previously described in mosquitoes from Thailand. Of particular significance, we identified viruses belonging to viral families (e.g. Our small RNA based strategy was also successful in characterizing viruses in published small RNA datasets from plants, fish and mammals in addition to insects. In the case of mouse samples, enrichment of viral sequences in the small RNA fraction was observed even in the absence of activation of the RNAi pathway. Thus, efficient production of virus-derived small RNAs might be a broad phenomenon that can be further explored for virus detection. Indeed, multiple mammalian antiviral pathways, including RNAi and RNase L, can generate small RNAs during viral infection ,56. HoweDatasets were deposited on the Small Read Archive of the National Center for Biotechnology Information under accession numbers described in Supplementary Table S2. Viral sequences described in Table Supplementary Data are available at NAR online.


Escherichia coli using RNA-seq, and present evidence that the stereotypical exponential RNA decay curve obtained using initiation inhibitor, rifampicin, consists of two phases: residual RNA synthesis, a delay in the interruption of steady state that is dependent on distance relative to the mRNA's 5′ end, and the exponential decay. This gives a more accurate RNA lifetime and RNA polymerase elongation rate simultaneously genome-wide. Transcripts typically have a single RNA decay constant along all positions, which is distinct between different operons, indicating that RNA stability is unlikely determined by local sequences. These measurements allowed us to establish a model for RNA processing involving co-transcriptional degradation, providing quantitative description of the macromolecular coordination in gene expression in bacteria on a system-wide level.An essential part of gene expression is the coordination of RNA synthesis and degradation, which occurs in the same cellular compartment in bacteria. Here, we report a genome-wide RNA degradation study in  The data were segmented into 300-nucleotide bins according to transcription unit annotation . We found that lifetimes were still generally constant across transcripts, and averaged ]4.5 min, with a smaller deviation than for exponential phase lifetimes  than in lifetimes (R2 = 0.05–0.13)  Fig and D.et al, We observed that some transcripts, like the smtA-mukFEB and hybOABCDEFG operons mentioned earlier, are so long that the amount of time needed to synthesize the full-length RNA is longer than the lifetime of the 5′ end of the transcript. In other words, the 5′ end is degraded before the 3′ end is synthesized, which is called co-transcriptional degradation. The degradation of the 5′ end of an RNA before the synthesis of its 3′ end has previously been shown for the trp and lac operons . We further assume that RNase that is bound to the transcript closely trails behind ribosomes, rapidly cleaving the ribonucleic acid after the last ribosome molecule, instead of being able to cut between ribosomes . This means that RNA abundance exhibits a position-dependent delay followed by single exponential decay at all segments of an RNA, similar to our experimental observations. This is in contrast to the post-transcriptional model, which has a more complex solution. It has a delay (equilibrium), followed by linear decay and then finally an exponential decay. Like in the co-transcriptional model, the delay is dependent on the distance of the segment from the 5′ end, while the linear decay is due to waiting for RNA synthesis to complete before degradation can commence (Supplementary Information).d of RNase is extremely high, RNA instantaneously degrades the moment it is completely synthesized, the degradation of the RNA abundance at the 5′ end is exactly linear (Supplementary Information). When the binding rate d is finite, the decay curve of the RNA abundance at the 5′ end should always be slower than that of instantaneous degradation. Therefore, in such a post-transcriptional case, the RNA lifetime at the 5′ end should be greater than the synthesis time for the whole transcript.In the fastest case of post-transcriptional degradation, that is, the binding rate On the contrary, when the ratio of synthesis time/lifetime > 1, there are moments where the 5′ end RNA abundance is lower than that expected from the fastest post-transcriptional decay mechanism, for example, the smtA-mukFEB operon . It indiet al, dL/v (Supplementary Information), which is dependent on the ratio of RNA synthesis time to RNA lifetime. For transcripts with a synthesis time/lifetime (dL/V) > 1, the probability of each RNA molecule being co-transcriptionally degraded is > 60%. If the co-transcriptional degradation model holds genome-wide, even for transcripts that have a synthesis time/lifetime < 1, we can still calculate that a sizeable fraction of RNA that is simultaneously being synthesized and degraded. For instance, with a synthesis time/lifetime of 0.5, 40% of RNAs are degraded before completely being synthesized.We caution that a co-transcriptional degradation mechanism does not imply that every single produced RNA in the cell is being synthesized and degraded simultaneously. Both ribosome binding, which is proposed to be protective  were grown in LB-Miller (EMD) at 30°C with shaking at 250 rpm. Overnight cultures were diluted 1:250 into fresh media and grown to OD600 = 0.3 for the exponential phase sample and OD600 = 1.7 for the stationary phase sample. The doubling time during steady-state growth in exponential phase was ]40 min.Rifampicin , dissolved in DMSO, was added to MG1655 and AS19 cultures at a final concentration of 500 ng/μl, and aliquots of the cultures were quenched in 10% volume of cold 9:1 ethanol:phenol solution at specified time points. The cells were pelleted and washed with cold PBS before storage at −80°C.Streptolydigin (ChemCon GmbH) was dissolved in water and added to AS19 cultures at a final concentration of 100 ng/μl.Bacterial pellets were thawed on ice. Cells were first resuspended and incubated in 1 mg/ml of lysozyme (Sigma) prepared in T.E. buffer and then completely lysed by the addition of 1 volume cell lysis buffer (Puregene). Spike-in RNAs were introduced at this stage.The samples were extracted with acidic phenol/chloroform (Sigma) 2–4 times and then once with chloroform:isoamyl alcohol . The RNA was then precipitated with the addition of 1/10 volume 5 M NaCl and 1 volume isopropanol on ice or in −20°C. RNA was pelleted and washed twice with 70% ethanol, resuspended to 100–200 ng/μl in nuclease-free water (Ambion or Cellgro), then digested with DNaseI (NEB) to remove contaminating DNA. The RNA was then purified as described. After resuspending in water, the RNA was used immediately.rRNA was removed by Epicentre's Ribo-Zero Kit (for gram-negative bacteria) following manufacturer's protocol. RNA was finally collected by RNA Clean-up and Concentration column (Zymo Research).The RNA was fragmented and prepared for Illumina sequencing as previously reported. The concentration of the libraries was determined by qPCR  according to Illumina's protocol, substituting DyNamo HS qPCR mix  for the Kappa qPCR mix. Samples were run on an updated Genome Analyzer II, or a HiSeq2000. The raw data are available from NCBI Short Reads Archive (BioProject PRJNA258398).et al, x is time (min), Pi is the polymerase passage time (delay) for fragment i, and Ti is the lifetime of fragment i. Elongation rates were fitted linearly using Pi for each fragment by a custom Igor program. To ensure proper fitting, only transcripts longer than 1,200 nt (giving 4 data points) were analyzed. Transcription units with large uncertainty in fitting (errorrate/rate > 1) or nonsensical results (0 < errorrate/rate) were discarded. Other manipulations were performed in Excel. The relationship between rRNA chain elongation rate and culture temperature is known  using Bowtie 0.12.7. The mapped reads were then collated and normalized across time points by the average of the total number of spike-in reads, ssrA, ssrS, and rnpB, before normalizing by abundance at time 0 min. Within transcription units, the data were broken into 300-nucleotide (nt) bins. Transcription units that have overlapping annotations were not included in the dataset (Keseler


Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.Dendritic cells (DC) play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA) carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both  Dendritic Cells (DCs) play crucial roles in promoting and regulating immune responses, including adaptive immune defences. An important aspect is their capacity for handling antigens for delivery and presentation to the adaptive immune defence compartments . AdvanceAlthough there are reports claiming targeting of DCs, many of these do not actually study interaction with the cells, presuming that an induced immune response reflected DC delivery. While this may be the case, it is not guaranteed for nucleic acid delivery. DNA vaccines can be delivered to promote immune response development, but the process of DNA nuclear translocation would not provide the efficient means of RNA delivery. An important property offered by nucleic acid vaccines relates to the initiation of potent immune responses benefitting from vaccines resembling the natural infection of the pathogen in question ,4. This The DCs therefore offer valuable targets for RNA vaccine delivery, being so central to promoting immune response development. Accordingly, this review will discuss how recent advances in RNA and biocompatible delivery vehicle technologies are advancing RNA vaccine development. The main aims focus on nanoparticle structures enhancing DC interaction, the consideration of cellular endocytic pathways for determining the outcome of vaccine delivery, and the application of self-amplifying replicon RNA (RepRNA) vaccines. As with mRNA vaccines, RepRNA vaccines require appropriate cytosolic delivery and transfer to the site of translation. Of particular importance are the roles of targeting ligands influencing delivery and intracellular compartmentalization, and cationic elements for cytosolic translocation. The high potential of the RepRNA delivery for vaccine applications will be elaborated in this review, proposing how progress will lead to enhanced self-amplifying vaccine targeting to DCs.RepRNA is derived from defective virus genomes . While RWhen RNA is delivered to DCs, it is necessary that it reach the translation machinery to translate its encoded antigens, as well as its polymerase proteins for replication of a RepRNA ,7,8,9,10in vitro prior to their transfer into vaccines [A major problem for RepRNA application has been its high RNase sensitivity, for which reason delivery has employed virus-like particle vectors: virus-like replicon particles (VRPs) ,9,10,22.vaccines ; unfortuAn alternative to VRP-based delivery of RepRNA vaccines is application of biodegradable nanoparticulate vehicles , which h+ or Lamp1+ (CD107a) late endocytic compartments, for release in a small Rab27-dependent manner. Interestingly, similar late endosomal structures under Rab27 control are involved in processing for the MHC Class II presentation to Th-lymphocytes. This demonstrates that the DC endocytic pathways do not necessarily lead to protein antigen degradation. The cross-presentation pathways also employ routes involving endosomal escape, leading ultimately to the proteosome for processing and association with MHC Class I molecules in the endoplasmic reticulum (ER) [Appropriate targeting of protein-based vaccines to DC can promote processing of the antigen through the DC endosomal system for presenting derived peptides in association with MHC Class II molecules to Th-lymphocytes ,36,37,38lum (ER) ,43,44,45+ lamina propria DC apparently arise from the same lineage and pre-cDC as blood cDC; CX3CR1+ lamina propria DC arise from the same lineage (in the mouse) as monocytes and inflammatory macrophages; Langerhans cells may also arise from this linage. Regulatory DC subsets also exist, particularly at mucosal surfaces, or induced by their environment. Targeting the appropriate DC subsets is crucial for efficient vaccine delivery leading to adaptive immune response development. While the receptors displayed on DC subsets have a strong influence, this is not merely in terms of targeting vaccine interaction with the appropriate subset, but also the consequence of that interaction in terms of the endocytic pathways employed by the DCs.Efficient induction of lymphocyte function following vaccination requires vaccine interaction with the conventional DC (cDC) subset , referreVaccine delivery to DCs will encounter the high capacity of DCs for internalizing a wide variety of antigen forms and types, through their diverse network of endocytic routes ,49,50,51+-ATPases necessary for gradual acidification of the endocytic vesicle in which the antigen is found, as well as the proteases  necessary for the processing. These proteases (cathepsins) are pH-sensitive; hence the gradual acidification to pH 5.5 as the endosome matures from an “early endosome” to a “late endosome”. An important consequence therein for RNA delivery is the escape of the RNA from this acidifying system prior to attack by the RNases. In this context, the action of the vacuolar H+-ATPases plays an important role, in a similar fashion to its role in initiation of the cross presentation pathways for antigen.There are certain crucial requirements involved in efficient processing of antigen, which also have a major bearing on RNA delivery and the likelihood of its translation. The endocytic processing pathways have been widely studied in the context of antigen presentation to Th lymphocytes ,37,38. TAs with DC processing of antigen for presentation to Th lymphocytes, both exogenous and endogenous antigen can be processed for Tc lymphocytes. This processing of exogenous material via “cross-presentation” pathways ,43,44,45An important issue pertinent to nanoparticle delivery is the size of the material endocytosed; this influences both the endocytic route of uptake and the consequences on cell handling of the endocytosed material ,44. Smalet al. [It would appear that the majority of endocytosed material reaching macropinosomes accumulates in lysosomes ,49,50,51et al.  proposedMany vaccines in current use are non-replicating entities. Although nanoparticle delivery vehicles have been widely applied in vaccinology, a major focus has been humoral immunity; indeed, the capacity for promoting the various arms of immune defence may prove limited. Replicating vaccines by their nature may provide conditions more related to when immune defences develop following pathogen infection; thus, a greater capacity for inducing both humoral and cellular (cytotoxic) immune defences is conceivable. Such vaccines cannot be created for all pathogens, and live vaccines present a risk of reversion to more pathogenic progeny.While application of DNA vaccines has received much attention, the DC nuclear membrane can prove quite resistant to nucleic acid translocation ,56. This+-ATPases. A major difference among the different routes is the rate at which this occurs, clathrin-mediated endocytosis tending to appear the more rapid [While many studies on DC endocytosis have focused on clathrin-mediated uptake , the rapre rapid ,49,50,51An important consequence for successful RNA vaccine delivery must be cytosolic translocation from the endocytic vesicle to permit interaction with the cellular translation machinery. Such cytosolic delivery can be enhanced by modifying the consequences of endosomal acidification on the endocytic vesicle integrity ,68. When+-ATPases pump protons into the acidifying endocytic vesicle, such nanoparticulate structures can provide the so-called “proton sponge effect” [It is considered that structures such as chitosan and PEI containing protonable amines  can buff effect” . Certain effect” ; histidi effect” ,68. The The proton sponge effect is not the only manner by which cytosolic translocation may be promoted. Interaction with the anionic vesicular membrane is an important consideration , potentiAlthough delivery of oligonucleotides for RNA interference therapy have successfully employed cationic-based delivery vehicles ,61,67,75With self-amplifying RepRNA offering high potential in vaccinology due to its replicative nature , criticain vivo [in vivo [The potential for biocompatible nanoparticulate vehicle delivery of RepRNA to DCs was proposed as early as 2008  Figure ; followiin vivo . However[in vivo . RepRNA [in vivo  for dest[in vivo ,68,74,85[in vivo , which h[in vivo .This article also confirmed that differences could be observed for RepRNA and oligoRNA delivery . Not onlin vivo for inducing immune responses, observed in both mice and rabbits [h-lymphocyte and Tc-lymphocyte immunity were identified. These responses were monitored with reference to anti-HA and anti-NP activity induced by the influenza virus HA and NP encoded by the RepRNA. Interestingly the authors employed two RepRNA molecules, each encoding one of the influenza virus antigens, and each encapsulated individually. Vaccination employed an equimolar mixture of the two formulated RepRNA, which proved to be a clearly successful approach. An additional report using an alphavirus replicon with in vivo liposome-like delivery [in vivo. Such an observation cannot be repeated with an RNase-sensitive pestivirus RepRNA, which does not lead to translation or induction of immune responses in vitro or in vivo when employed as “naked” RNA [et al. [The observed successful delivery of pestivirus RepRNA to DCs provided the necessary RepRNA translation  rabbits . When thdelivery  was alsoked” RNA . These oked” RNA ,91,92,93ked” RNA . The 5' ked” RNA ,96 and tked” RNA ), althouked” RNA ,98; Ross [et al. . It woul [et al. ,86, rela [et al. ,91,92,93 [et al. . Such vi [et al. ,100. Thu [et al. .in vivo is achievable by complexing with BSA [It has also been reported that successful RNA delivery with BSA . Employiwith BSA . Virionswith BSA ; again nin vivo work [The recent work on NGA-delivery of cap-independent RepRNA to DCs  has demoivo work  confirmsin vivo. For both humoral immunity and CMI, the inherent characteristics of the DCs for handling antigen can be employed by targeting the RepRNA vaccine to the cell type ensuring efficient immune defence induction. In this context, an important consideration is the application of a RepRNA non-cytopathic for the DCs. One aspect of this delivery to DCs that can be enhanced is the targeting element. Current work is pursuing this aspect to determine how the characteristics of targeting relate to particular endocytic processing and ultimately how this impacts on the efficiency of the RepRNA translation and replicon, leading to induction of both the humoral and cytotoxic arms of immune defense.To date, synthetic particle delivery of RNA has focused on siRNA and mRNA. There were no reports on RepRNA interaction with DCs until recently . This wo


Caenorhabditis elegans. Both genetically silencing and ChR2-mediated activation of ADL sensory neurons significantly affected preference choice. ADL regulated preference choice by inhibiting function of G protein-coupled receptor (GPCR)/SRH-220. ADL sensory neurons might regulate preference choice through peptidergic signals of FLP-4 and NLP-10, and function of FLP-4 or NLP-10 in regulating preference choice was regulated by SRH-220. FLP-4 released from ADL sensory neurons further regulated preference choice through its receptor of NPR-4 in AIB interneurons. In AIB interneurons, NPR-4 was involved in the control of preference choice by activating the functions of ASH-2 trithorax complex consisting of SET-2, ASH-2, and WDR-5, implying the crucial role of molecular machinery of trimethylation of histone H3K4 in the preference choice control. The identified novel neuronal circuit and the underlying molecular mechanisms will strengthen our understanding neuronal basis of preference choice in animals.Preference choice on food is an important response strategy for animals living in the environment. Using assay system of preference choice on bacterial foods, OP50 and PA14, we identified the involvement of ADL sensory neurons in the control of preference choice in  Caenorhabditis elegans is an attractive model animal for behavioral study due to its simple nervous system and ability to sense diverse environmental stimuli, such as touch, smell, taste and temperature3C. elegans nervous system makes it a useful assay system for seeking the biological mechanisms of behavioral choice5C. elegans can exhibit several types of behavioral choice based on its response to paired or multiple stimuli, such as attractants versus aversive stimuli or different attractants7891011Animals’ decision making includes two main levels of complexity, behavioral choice and value-based decision making1C. elegans, olfactory chemotaxis towards food-associated odors is an innate behavior, and highly reproducible among animalsC. elegans121212C. elegans, AWB and AWC sensory neurons are necessary for innate preference for Pseudomonas aeruginosaC. elegans.In C. elegans, ASH and ADL are important sensory neurons functioning to sense and mediate avoidance of noxious environmental stimuli, such as Cu2+ ion and garlic1819C. elegans. The new identified neuronal circuit and the related underlying mechanism for preference choice on bacterial food will strengthen our understanding neuronal basis of behavioral choice in animals.Among the well-known 12 kinds of amphid neurons  in egl-1 gene encodes a cell-death activator , and can be used to genetically ablating specific neuronsegl-1 under the control of srh-220 promoter. Interestingly, genetically ablating ASH sensory neurons did not significantly affect preference choice compared with wild-type N2  mutant had the similar preference choice to wild-type (data not shown). We observed that nematodes with channelrhodopsin-2 (ChR2)-mediated activation of ADL sensory neurons on assay plates with all-trans retinal (ATR) exhibited significantly enhanced choice index compared with wild-type N2  mutant had the similar leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to wild-type (data not shown). Meanwhile, nematodes with optogenetically activating ADL sensory neurons showed the similar leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 compared with wild-type N2  adaptor protein, and plays important roles in both longevity and immunityarr-1 gene caused the deficit in preference choice behavior compared with wild-type N2 (arr-1(ok401) mutant nematodes had similar leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to those in wild-type N2 (arr-1 gene under the control of srh-220 promoter rescued the deficit in preference choice in arr-1(ok401) mutant (In -type N2 . arr-1(o-type N2 . Moreove) mutant , suggestC. elegans, some genes encoding GPCRs such as SRE-1, SRI-51, SRH-132, and SRH-220 are expressed in ADL sensory neurons2425srh-220 gene resulted in enhanced choice index compared with wild-type N2 (srh-220(tm3939) mutants had normal leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 (srh-220(tm3939) could be rescued by the expression of srh-220 under the control of its own promoter  releaseunc-31 gene significantly decreased choice index compared with wild-type N2  is a gain-of-function mutant, and pde-4(ok1290) is a loss-of-function mutant. We further found that the gsa-1(ce94) mutant had the increased choice index, whereas the pde-4(ok1290) mutant has the decreased choice index compared with wild-type N2  mutants had normal leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 (In -type N2 . Nematod to OP50 . Therefosrh-220 and flp-4 or nlp-10 in regulating preference choice in nematodes. We found that the preference choice phenotype in double mutant of flp-4(RNAi);srh-220(tm3783) was similar to that in flp-4(RNAi) nematodes (nlp-10(tm6232);srh-220(tm3783) was similar to that in nlp-10(tm6232) mutant nematodes  mutants had normal leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50  mutants (6npr-4(tm1782) mutants . Among 24 genes expressed in AIB interneurons, we found that mutation of npr-4 gene caused the significant increase in expression levels of ptp-3, and ced-10 genes, and the significant decrease in expression levels of glr-2, tax-6, cdc-42, and set-2 genes  methyltransferase.To identify candidate targeted genes for -2 genes . In C. enpr-4 to investigate their possible function in regulating preference choice in nematodes. Among 7 candidate genes examined, mutation of set-2 gene caused the significant decrease in choice index compared with wild-type N2 (set-2(ok952);npr-4(tm1782) was similar to that of single mutant of set-2(ok952) or npr-4(tm1782) (set-2(ok952) mutants had similar phenotypes of leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to those in wild-type N2 (We further used the available mutants for candidate targeted genes of -type N2 . Moreove(tm1782) , suggest-type N2 .C. elegans, it was reported that ASH, WDR-5, and SET-2 constitute an ASH-2 trithorax complex, which trimethylates histone H3K4ash-2 or wdr-5 gene caused the significant decrease in choice index compared with wild-type N2; however, mutation of rbr-2 did not significantly affect preference choice behavior (In behavior . These rash-2(tm1726) or wdr-5(ok1417) mutants had similar phenotypes of leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to those in wild-type N2  mutants  X, arr-1(ok401) X, srh-220(tm3939) IV, flp-21(ok889) V, nlp-7(tm1970) X, nlp-8(ok1799) I, nlp-10(tm6232) III, npr-4(tm1782) X, tax-6(ok2065) IV, glr-2(ok2342) III, ced-10(n1993) IV, ptp-3(ok244) II, cdc-42(ok825) II, gsa-1(ce94) I, pde-4(ok1290) II, set-2(ok952) III, ash-2(tm1726) II, wdr-5.1(ok1417) III, and rbr-2(ok2544) IV, and transgenic strains of quEx128[npr-9::GFP + pRF4 rol-6(su1006)] expressing npr-9 in AIB interneurons and ASH::TeTxNematodes were grown on nematode growth medium (NGM) plates seeded with E. coli OP50 and P. aeruginosa PA14. E. coli and P. aeruginosa cultures were grown in Luria-Bertani (LB) broth overnight at 37 °C.Bacterial strains used were E. coli OP50. All the behavioral experiments were performed with young adults maintained at 20 °C. Behavioral choice assay was performed on 9-cm NGM plates. Bacterial food with a diameter of 0.5-cm was seeded at the distance of 1.5 cm from periphery /total number. Ten replicates were performed.C. elegans genomic DNA  was inserted into 95_77-mcherry vector and then transformed into the strain of quEx128. Germline transformation was performed as describeddop-1::rfp or Plin-44::gfp at a concentration of 60 μg/mL into the gonad of nematodes.To generate entry vectors carrying promoter sequences, promoter regions were amplified by polymerase chain reaction (PCR) from wild-type Chlamydomonas reinhardtii, can utilize chromophore ATR to enable the activity of specific neurons in C. eleganssrh-220 promoter was used for the ADL-specific expression, and the npr-9 promoter was used for the AIB-specific expression. Nematodes expressing ChR2 in ADL sensory neurons or AIB interneurons were grown on OP50-seeded NGM agar plates containing 50 μM of ATR. Plates were seeded on day 0. Nematodes were transferred to OP50-seeded plates containing 50 μM of ATR on day 1 in the dark. After starvation for 5 h, nematodes were transferred to assay plates containing 50 μM of ATR for behavioral analysis on day 2. During the assay, whole field illumination was performed, and ChR2 was excited by a round blue light in diameter of 9.5 cm sourced from LED array  constructed in a LED light source . Light intensity measured at the sample was 5 mW/mm2 of 465 nm light. For optogenetic experiments, light intensity was monitored using an optical power meter . All work was carried out under low-illumination conditions to prevent the preactivation of ChR2-expressing neurons. Nematodes without ATR treatment were used as the control. Optogenetical activation was performed under the lite-1 mutation background. Ten replicates were performed.Light-activated ChR2, a directly light-gated cation channel from the green alga E. coli strain HT115 (DE3) expressing double-stranded RNA that is homologous to a target gene as describedE. coli HT115 (DE3) grown in LB broth containing ampicillin (100 μg/ml) at 37 °C overnight was plated onto NGM containing 100 μg/mL ampicillin and 5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). L2 larvae were placed on RNAi or vector control plates for 2 days at 20 °C until nematodes became gravid. Gravid adults were transferred to fresh RNAi-expressing bacterial lawns and allowed to lay eggs for 2 h to obtain the second generation of RNAi population. Eggs were allowed to develop at 20 °C to young adults for the subsequent assays. unc-22 RNAi was included as a positive control.RNAi was performed by feeding nematodes with flp-4, pde-4, unc-31, or gsa-1 gene was amplified by PCR to yield product A. The srh-220 promoter was used for ADL-specific expression, and amplified in two different reactions with promoter reverse sense primer or promoter reverse antisense primer together with promoter forward primer to yield product B and C. Product A was further fused by amplification to B or C using the nested primers to yield fragments D and E. In fragment D, the flp-4, pde-4, unc-31, or gsa-1 gene was transcribed by ADL-specific promoter in the sense orientation. In fragment E, the flp-4, pde-4, unc-31, or gsa-1 gene was transcribed by the ADL-specific promoter in the antisense orientation. The fragments D and E were mixed in equimolar amounts and injected at 25–100 ng/μL into the wild-type nematodes. The Plin-44::gfp was used as a transgenic marker.The method was performed basically as describedact-1 gene was chosen as a reference gene. Relative quantification of targeted genes in comparison to reference act-1 gene was determined, and the final results were expressed as relative expression ratio between targeted gene and reference gene. The primer information was shown in Total RNA of nematodes was extracted using RNeasy Mini Kit . Purity and concentration of RNA were evaluated by OD260/280 in a spectrophotometer. Total RNA was reverse-transcribed using cDNA synthesis kit . After cDNA synthesis, real-time PCR was performed using SYBR Premix Ex Taq™ (Takara) for amplification of the PCR products. The All data in this article were expressed as means ± standard error of the mean (S.E.M.). Graphs were generated using Microsoft Excel . Statistical analysis was performed using SPSS 12.0 . Differences between groups were determined using analysis of variance (ANOVA). The probability levels of 0.05 and 0.01 were considered to be statistically significant.How to cite this article: Yu, Y. et al. FLP-4 neuropeptide and its receptor in a neuronal circuit regulate preference choice through functions of ASH-2 trithorax complex in Caenorhabditis elegans. Sci. Rep.6, 21485; doi: 10.1038/srep21485 (2016).


A growing number of middle-income countries are scaling up youth-friendly sexual and reproductive health pilot projects to national level programmes. Yet, there are few case studies on successful national level scale-up of such programmes. Estonia is an excellent example of scale-up of a small grassroots adolescent sexual and reproductive health initiative to a national programme, which most likely contributed to improved adolescent sexual and reproductive health outcomes. This study; 1) documents the scale-up process of the Estonian youth clinic network 1991–2013, and 2) analyses factors that contributed to the successful scale-up. This research provides policy makers and programme managers with new insights to success factors of the scale-up, that can be used to support planning, implementation and scale-up of adolescent sexual and reproductive health programmes in other countries.Information on the scale-up process and success factors were collected by conducting a literature review and interviewing key stakeholders. The findings were analysed using the WHO-ExpandNet framework, which provides a step-by-step process approach for design, implementation and assessment of the results of scaling-up health innovations.The scale-up was divided into two main phases: 1) planning the scale-up strategy 1991–1995 and 2) managing the scaling-up 1996–2013. The planning phase analysed innovation, user organizations (youth clinics), environment and resource team . The managing phase examines strategic choices, advocacy, organization, resource mobilization, monitoring and evaluation, strategic planning and management of the scale-up.The main factors that contributed to the successful scale-up in Estonia were: 1) favourable social and political climate, 2) clear demonstrated need for the adolescent services, 3) a national professional organization that advocated, coordinated and represented the youth clinics, 4) enthusiasm and dedication of personnel, 5) acceptance by user organizations and 6) sustainable funding through the national health insurance system. Finally, the measurement and recognition of the remarkable improvement of adolescent SRH outcomes in Estonia would not have been possible without development of good reporting and monitoring systems, and many studies and international publications. A small but growing number of middle-income countries are scaling up pilot projects on youth-friendly sexual and reproductive health (SRH) services, to district, province and national level programmes. Yet, there are few case studies or publications on successful national level scale-up of such programmes. This study: 1) documents the scale-up process of the Estonian Youth Clinic Network (YCN) during the period 1991–2013, and 2) analyses factors that contributed to the successful scale-up of the YCN, by using the WHO-ExpandNet framework.Estonia is the northernmost of the three Baltic States. The population is 1.3 million, of which 12%  are 15–24 years[Estonia is an excellent example of scaling up a small grassroots SRH initiative to a national YCN, which most likely contributed to improved adolescent SRH outcomes. The first youth clinic (YC) was started in 1991. During the following decade the YCN was successfully scaled up to a national network of 18 YCs, which is coordinated and represented by the Estonian Sexual Health Association (ESHA). SRH outcomes of Estonian youth improved remarkably during the period 2001–2009. Annual rates of abortions, STIs and diagnosed HIV infections among 15–24 year olds were reduced by 37%, 55% and 89%, respectively, 7. The A systematic search was conducted to identify relevant literature on the Estonian YCs. A snowball sampling technique, i.e. by reviewing references and other publications of selected authors was used to identify more publications, including grey literature. The initial literature review showed that most publications were written from the perspective of the YCN. Therefore, additional information was collected from two complementary perspectives by interviewing: 1) Dr Tiiu Aro, a former Minister of Social Affairs of Estonia (MSA) 1996–1999, who provided a policy makers point of view, and 2) Dr Sirje Vaask who gave insights in the development of the Estonian Health Insurance Fund (EHIF) and its relationship with the YCs. Moreover, the authors were already familiar with the YCN through their previous work.The scale-up process and success factors of the Estonian YCN were analysed with the WHO-ExpandNet framework, which provides a step-by-step process approach for design, implementation and assessment of the results of scaling-up health innovations. It contThe findings are presented in two main phases: 1) planning the scale-up strategy 1991–1995 and 2) managing the scaling-up 1996–2013. In 1996 the pace of establishing new YCs accelerated. Therefore this year was chosen as a starting point of the scale-up phase. The timeline in Figure The planning strategy section covers four key elements: 1) innovation, 2) user organizations, 3) environment, and 4) resource team.innovation refers to health service components or practices that are new or perceived as new in a particular context[adoption of the Swedish model of delivering youth-friendly SRH services in specialized YCs. According the framework attributes of effective innovations are; credibility, relevance, clarity, compatibility and ease of installation.In the WHO-ExpandNet framework  context. In thiscredibility of this innovation. In this model SRH services, that are confidential and free of charge, are delivered in a youth-friendly atmosphere in specialized YCs. Personnel of the YCs are trained in youth-friendly SRH services.Sweden has had a national system of YCs for several decades, which added to the Adoption of the innovation was a response to the lack of specialized youth-friendly SRH services in post-Soviet Estonia. The innovation targeted the high rates of unwanted pregnancy, abortion and STIs among young people in the early 1990s[The YCs provide SRH services to young people up to 24 years. The YCs provide also sexuality education lessons in their premises and in schools.The new youth-friendly SRH services were supplementary to the existing adult focused services and provided (mainly) within the same premises.The first YC, founded in Viljandi in 1991, was still a small local initiative, which provided only a few hours of youth-friendly SRH services per week. In the following four years four more YCs were founded in Tallinn, Tartu, Haapsalu and Paide. The SRH service package developed gradually when more YCs were established.user organizations refer to the institutions that adopt and implement the innovation[In the WHO-ExpandNet framework novation. In the novation. The twoAccording to the framework the attributes of effective user organizations are; commitment, credibility and capacity.The need for the adolescent SRH services was recognized by the user organizations, because the early initiatives came from persons who worked for or with the user organizations. Moreover, new epidemiological data and comparison with neighbouring Nordic countries demonstrated the need to improve SRH of Estonian adolescents. The useThe new youth-friendly services were implemented mostly within existing healthcare facilities by specialized (re-)trained personnel. Moreover, in hospitals and private practices the new youth-friendly SRH services were supplementary to the existing adult focused services, which eased the cooperation. The innovation was rolled out on healthcare facility level. Therefore decision making and improving implementation capacity, i.e. personnel training and space, were done locally.environment refers to conditions which are external to the user organization, but fundamentally affect the prospects for scaling up[In the WHO-ExpandNet framework aling up. The maiThere was a favourable social and political climate for implementation of the YCN in post-independence Estonia. Unlike in many other countries, there was only moderate moral, religious or political opposition to adolescent SRH interventions.Major political and economic changes took place in Estonia in the early 1990s. The country was transformed from a centrally controlled communist system to a modern democratic and free market society. The collapse of the Soviet Union caused a deep recession in Estonia, which limited funding of the healthcare system. Consequently, the main challenges for the YC scale-up were financial.The post-independence period was characterized by aspirations for a better future and eagerness to learn from experiences and best practices abroad and implement them locally. This was essential for initiation of new international cooperation and seizing opportunities to introduce youth-friendly SRH interventions, which had been proven successful abroad. This included adoption of the Swedish model of YCs.In the early 1990s adolescent SRH outcomes were poor in Estonia. The main concerns at that time were: 1) low use of reliable contraceptive methods, 2) high unwanted pregnancy and abortion rates, and 3) sharply rising incidence of STIs, 11. It resource team refers to the individuals and organizations that seek to promote and facilitate wider use of the innovation[In the WHO-ExpandNet framework novation. In the According to the framework the attributes of effective resource organizations are; leadership, credibility and commitment.Estonian Sexual Health Association (ESHA) is a national NGO that coordinates and represents the independent YCs. Founding of ESHA in 1994 was a milestone in the expansion of the first YCs to a national YCN. ESHA has four employees and a workgroup which includes current and previous ESHA project managers, managers of the YCs and youth representatives.commitment of ESHA’s and YCs’ personnel was crucial for transforming the first grassroots initiatives to a national network. Through the years, ESHA became a credible partner for the government and EHIF.ESHA coordinated and monitored the YCs, developed quality standards, provided continuous training and lobbied for political commitment and funding. Moreover, Substantial international assistance was available for improvement of adolescent SRH in Estonia. Several international organizations; the International Planned Parenthood Federation European Network (IPPF-EN), WHO Regional Office for Europe, Swedish International Development Agency (SIDA) and the World Bank (WB) and Global Fund for AIDS, Tuberculosis and Malaria (GF), supported creation of the YCN. The involvement of these international agencies increased the credibility of the innovation.Support from IPPF-EN was particularly important. It assisted with the creation of ESHA and offered trainings, especially on management and advocacy. Moreover, it provided moral and some financial support. The memberships of IPPF-EN created an opportunity to network and cooperate with other SRH programmes in Finland, the Netherlands, Norway and Sweden.The neighbouring countries provided models, training, material and financial assistance. Adoption of the Swedish model required training of Estonian specialists. In the beginning of the 1990s several Estonian SRH pioneers, gynaecologist, midwifes and counsellors, made study visits to YCs in Sweden and participated in youth-friendly SRH service courses in the Netherlands. Moreover, training materials were adapted and translated to Estonian. The adoption process included also other projects, for example SIDA’s "From Abortion to Contraception" in 1993–1995.This managing the scale-up section has two subsections: 1) organization and management, which covers: strategic choices for scaling up, advocacy, organizing the scaling-up process, mobilization of resources, and monitoring and evaluation, and 2) strategic planning and management of scale-up.scaling up strategy refers to the plans and actions necessary to fully establish the innovation in policies and programmes. The YCN was scaled up by using a combination of strategies.In the WHO-ExpandNet framework the horizontal scaling up, which was done by replicating YCs to new geographic areas. ESHA’s horizontal expansion approach included; 1) engagement of local decision-makers, 2) assistance with establishment and organization of new YCs, 3) training of YCs’ personnel, 4) guidelines on quality requirements and operation principles for YCs[The primary strategy was  for YCs, and 5) Vertical scaling up of the YCN included institutionalization of the YCs and ESHA through: 1) ESHA and YC personnel’s’ lobby for political commitment and funding, 2) legislation change to establish EHIF[ish EHIF, and 3) spontaneous scaling up occurred, i.e. diffusion of YCs to new locations without deliberate guidance. Positive early experiences and media attention attracted enthusiasm of other SRH specialists. EHSA consolidated the spontaneous expansion by providing guidelines, training and assistance with founding and operating new YCs.Also some Advocacy was important for the successful scale-up. Personnel of ESHA and the YCs played an active role in advocating and promoting youth-friendly SRH services to policy makers and medical professionals. Repeated lobbying and fundraising efforts helped policymakers and EHIF directors to form a favourable opinion of the YCs. The need for youth SRH services was advocated by epidemiological data and many research results on adolescent SRH in Estonia. Between 1994 and 2007 there were 12 national surveys, which included adolescent sexual health items[th items. At the organizational approaches. It was a multiplicative process, where ESHA built the network together with new user organizations. The "recruitment" of new user organizations for the expansion was decentralized, while the financing mechanism of the YCN became centralized. The implementation itself was a standardized expert-led process. ESHA played a key role in defining and ensuring service quality. Well-defined objectives and quality requirements were important for standardization of the scale-up. EHSA prepared a practical guideline: Quality Requirements and Operational Principles for the YCN in 2002[The scale-up of the YCN included a combination of  in 2002, 16, 17.Mobilization of resources was essential for the successful scale-up of YNC. The Estonian healthcare reform project was implemented in 1995–1999. The World Bank lent MSA 18 million USD for the project[The deep recession in the early years of independence, was followed by 13 consecutive years of economic growth 1995–2007)[95–2007[Estonian Health Insurance Fund (EHIF)[In 2001 the regional sickness funds were reorganized into a single independent public agency, the d (EHIF). The newd (EHIF). This enSome adjustments were made on EHIF’s insurance coverage during the scale-up. New laboratory tests and rapid HIV tests were added to the package. Reimbursement rates of some services were increased. However, the economic crisis in 2009 forced EHIF to lower YCs’ reimbursement rates. Since then the total budget of the 18 YCs stabilized at approximately €950,000 per year .HIV incidence rose sharply in Estonia in the beginning of the 2000s[he 2000s, 23. Thehe 2000s. In respmonitoring were important for the successful scale-up of the YCN. The YCs report quarterly to ESHA. The primary use of the reporting and monitoring is management of the YCN. The information is also used for planning. The reporting includes: visitor data, medical tests and procedures, diagnosed STIs and pregnancies. Moreover, the YCs send monthly medical invoices to EHIF. The parallel reporting is used for reimbursement payments and monitoring annual YC budgets. Furthermore, ESHA reports quarterly to EHIF on activities of the YCN and services provided by the YCs.Regular reporting and evaluation of two YCs in 1997 encouraged further expansion of the youth-friendly SRH services[An external services. In 2008services. The resservices–29. MoreSRH service package remained mainly unchanged during the scale-up process. Several additions and improvements were made: 1) HIV testing and counselling services were introduced in response to the HIV epidemic in 2000, 2) the YCN was expanded to predominantly Russian speaking areas of Estonia (Ida-Virumaa region and Narva), 3) efforts were made to increase service uptake of young men, by providing separate visiting hours for them, 4) information dissemination on availability of YCs was improved, 5) personnel shortages were addressed, 6) larger YCs started to use online appointment scheduling systems, 7) reporting of the YCs was enhanced, and 8) a website and internet counselling services were launched first in Estonian and later in Russian.The core STI consultations; i) STI testing, treatment and follow-up consultations, ii) HIV services; voluntary confidential counselling and testing and in case of an HIV positive result referral for specialist consultation, and iii) HPV vaccination and counselling.Contraceptive consultations; i) information and counselling about contraceptives, ii) prescription and renewal of prescriptions for contraceptives, and iii) insertion of contraceptive devices.SRH counselling; i) psychosexual counselling , ii) pregnancy diagnostics and referral for antenatal care or safe abortion, iii) psychological counselling, and a range of iv) other SRH services, such as counselling for victims of sexual violence.Sexuality education lessons at schools or YCs.Website and internet counselling. On the website young people find information about puberty, intimate relationships, sexuality, pregnancy, contraceptives and STIs. The website also offers internet counselling services, where young persons’ anonymous questions about these topics are answered by personnel of the YCs.Currently the YCs provide the following SRH services:STI consimpact of the YCN. In the age group age 15–24 years the SRH outcomes improved remarkably during the period 2001–2009. Annual rates of abortions, STIs and diagnosed HIV infections in the age group were reduced by 37%, 55% and 89%, respectively[National age group specific SRH data provided a useful means of assessing ectively–33. The strategic planning was hindered by a continuous struggle for financing. The gradual growth was "organic" and built on short-term funding opportunities, which exposed the YCs to shifting local priorities. After 2002, when more sustainable EHIF funding became available, EHSA’s and YCs’ management teams could start to focus on long-term planning. At that time the YCN was already almost scaled up to the national level (15 YCs) and there was no need for strategic scale-up planning anymore. Therefore the attention was focused on management and professionalization of the YCN. Quality of services was standardized by providing guidelines, training and technical assistance. Client centred focus was maintained with continuous trainings and repeated client satisfaction surveys. Reporting and monitoring of YCs were enhanced. Improvements and adaptations were made to the service package. Vertical scaling-up efforts were continued by lobbying for sustainable funding and political support. During the scale-up process ESHA had proven to be a strong resource team and a credible partner for the YCs, the user organizations, EHIF and policy makers.In the early years of the scaling up YCN favourable social and political climate for offering youth-friendly SRH services. Major political and economic changes took place in Estonia in the early 1990s, which led to the transformation of the country from a centrally controlled communist system to a modern democratic and free market society. Unlike in many other countries, there was only moderate moral, religious or political opposition to the adolescent SRH services, which was successfully overcome by EHSA and its counterparts.Environment: Estonia had a need for the innovation was clearly demonstrated. In the early 1990s new epidemiological data demonstrated that adolescents SRH outcomes were much better in neighbouring Nordic countries. In response to this, the successful Swedish model of delivering youth-friendly SRH services in specialized YCs was adopted in Estonia.Innovation: The professional organization that coordinates and represents the YCs. The creation of ESHA in 1994 was a milestone in the scale-up process. ESHA also monitored the YCs, developed quality standards, provided continuous training and lobbied for political commitment and funding. Moreover, the enthusiasm and dedication of ESHA’s and YCs’ personnel were crucial for transforming the first grassroots initiatives into a national network. Through the years, ESHA became a credible partner for MSA and EHIF.Resource team: There is a national What were the main factors that contributed to the successful scale-up of the YCN in Estonia? The analysis of the scale up using the WHO-ExpandNet framework highlighted the following factors:international assistance was available for improvement of adolescent SRH in Estonia. Assistance from the neighbouring countries, i.e. providing models, training and practical and financial assistance was crucial for the development and scale-up of the YCN. This also applies to international organizations: WHO, WB, SIDA, GF and IPPF-EN. Support from IPPF-EN was particularly important. It assisted with the creation of ESHA and offered trainings, especially on management and advocacy. Further, the membership of IPPF-EN created an opportunity to network and cooperate with other SRH programmes in other countries.need for the adolescent SRH services was recognized by the user organizations , as the early initiatives came from persons who worked for or with the user organizations. Moreover, in hospitals and private practices the new youth-friendly SRH services were supplementary to the existing services, which made the scale-up easier.User organizations: The advocating and promoting youth-friendly SRH services to policy makers and medical professionals. The repeated lobbying and fundraising efforts helped policymakers and EHIF directors to form a favourable opinion of the YCs. The need for youth SRH services was advocated with epidemiological data and evaluation/research results on adolescent SRH.Advocacy: Personnel of ESHA and the YCs played an important active role in sustainable national funding from EHIF. A main turning point of the YCN was the 2002 legislation change and reorganization of health insurance system, which enabled EHIF to start to centrally finance the YCs. The stable funding environment allowed ESHA and the YCs to switch their focus from short-term financial survival to long-term planning, quality improvement and sustainable scale-up of the SRH services. In addition, the economic growth in Estonia certainly influenced the sustainability of YCs’ funding.Mobilization of resources: The YCs eventually received SRH outcomes among adolescents improved remarkably during the period 2001–2009. The YCN was developed simultaneously with the Estonian school-based sexuality education programme. Together the two interventions have most likely contributed to the improvement of SRH outcomes. Measurement of the improvement would not have been possible without development of good reporting and monitoring systems. The SRH improvements have been studied and documented in many studies and international publications on adolescent SRH in Estonia. The success of the Estonian YCN is nationally and internationally recognized.Recognition of the success: Finally, In addition, substantial This framework analysis has some limitations. Firstly, this is a retrospective assessment of the scale-up process by using the WHO-ExpandNet framework. The framework was not used in planning or management of the YCN scale-up in Estonia. Secondly, in practice the planning and managing scale-up phases used in the framework analysis overlapped and were not clearly defined. Thirdly, the first developments of the YCN took place 24 years ago. At times it was challenging to find documentation or reliable recollections of the early developments.The main factors that contributed to the successful scale-up of youth-friendly sexual and reproductive health services in Estonia were: favourable social and political climate, clear demonstrated need for the adolescent services, a national professional organization that advocated, coordinated and represented the youth clinics, enthusiasm and dedication of personnel, acceptance by user organizations  and sustainable funding through the national health insurance system. Finally, the measurement and recognition of the remarkable improvement of adolescent SRH outcomes would not have been possible without development of good reporting and monitoring systems, and many studies and international publications.


Background. Lack of adherence to dietary and physical activity guidelines has been linked to an increase in chronic diseases in the United States (US). The aim of this study was to assess the association of lifestyle behaviors with self-rated health (SRH). Methods. This cross-sectional study used self-reported data from Living for Health Program  which was conducted from 2008 to 2012 in 190 health fair events in South Florida, US. Results. Significantly higher percent of females as compared to males were classified as obese (35.4% versus 27.0%), reported poor/fair SRH (23.4% versus 15.0%), and were less physically active (33.9% versus 25.4%). Adjusted logistic regression models indicated that both females and males were more likely to report poor/fair SRH if they consumed ≤2 servings of fruits and vegetables per day  and consumed mostly high fat foods . The association of SRH with less physical activity was only significant in females . Conclusion. Gender differences in health behaviors should be considered in designing and monitoring lifestyle interventions to prevent cardiovascular diseases. Self-evaluation of general health status has been associated with actual health in that what people report about their health has been shown to predict mortality . Self-raHealth is largely influenced by modifiable risk factors such as diet and physical activity . NoncommLifestyle behaviors, particularly diet and physical activity, can contribute to or help prevent cardiovascular diseases such as coronary heart disease, hypertension, and type 2 diabetes . Diet anLack of physical activity and unhealthy dietary patterns place a burden on society due to lack of work productivity and increased health care costs . PhysicaN = 9,453). During these years, 190 health fair events were held in low income minority communities in Miami-Dade County to screen participants for cardiovascular disease risks. An accredited Institutional Review Board (IRB) provided a waiver of consent for the analyses. Self-rated health assessment was added in the year 2012 and was answered by 2,108 individuals during the remainder of the Living for Health Program. The final sample size  consisted of the participants who responded to all the questions included in this analysis.This is a secondary data analysis of the Living for Health Program . The Living for Health Program was conducted from 2008 to 2012 and collected information on health behaviors from adults ≥18 years old  excellent/very good/good or (2) fair/poor for this study. The sociodemographic questionnaire collected information on age, gender, ethnicity, smoking, health insurance, and comorbidities. Age was converted to a categorical variable  to assess differences between older and younger adults based on the demographics of the population. Smoking status derived from the question “do you smoke (any tobacco product)?” and was categorized as (yes/no). There were insufficient participants to access differences for the original five possible categories for ethnicity. Based on the distribution, ethnicity was collapsed into three categories: Hispanic, non-Hispanic Black, and other. Other category included White, American Indian, Pacific Islander, Indian, and Asian. Health insurance was categorized as a binary variable (no insurance/yes insurance). Presence of comorbidities (yes/no) was measured combining two questions: (1) “has any medical professional ever told you that you have high glucose, high blood pressure, high cholesterol, coronary heart disease)” and (2) “have you been prescribed medications for ?” A positive response to either question was categorized as having comorbidities.2). BMI was classified as BMI: 18.5–29.9 m2 (nonobese) and BMI: ≥30 (obese). There were <1.0% of participants classified as underweight (BMI <18.5); these participants were included in as part of the “nonobese” group.Participant's height and weight were measured and body mass index (BMI) was calculated as weight (kg)/height . The five possible options were 5 or more per day, 4 a day, 3 a day, 2 a day, or 1 a day. The responses were categorized into three as follows: (1) 5 or more per day; (2) 3-4 per day; (3) 2 or less per day.Fat intake was measured by the question: “if you ate fast food in the last month, what type was it?” Examples of high fat foods  and low fat foods , single burgers, grilled chicken, fruits, and yogurt parfaits) were provided. From the five possible options, the responses were categorized into three as follows: (1) primarily low fat foods, mostly low fat or some high fat; (2) both high fat and low fat foods about the same; (3) mostly high fat, some low fat or primarily high fat foods.Physical activity was measured with the question: “in the last month, how often were you physically active?” . Physical activity was collapsed from six possible options into three categories as follows: (1) 5–7 days a week; (2) 1–4 days a week; (3) 1–3 times a month or never to allow sufficient number in each category.P < 0.05. All statistical analyses were performed using IBM Statistical Package for the Social Science version 19 .The general characteristics of the participants were examined with descriptive statistics. Differences between male and female participants were assessed with the Chi-square test for categorical variables. Logistic regression models were conducted to examine the relationship between the binary dependent variable, SRH with fruits and vegetables intake, physical activity, and fat intake adjusting for confounding variables. Covariates were considered potential confounders based on the literature. Age and gender differences were found in perceptions of SRH , 29. PooP = 0.001), reported poor/fair SRH , and were less physically active . Participants who reported poor/fair SRH as compared to those with excellent/very good/good were females ; older > 55 years ; obese ; non-Hispanic Black ; had comorbidities ; consumed ≤2 servings of fruits and vegetables per day ; had an intake of primary high fat foods/mostly high fat or some low fat foods ; and were less physically active  (< 0.001) .P = 0.001, 95% CI 1.39–3.70). Logistic regression model adjusted for age, BMI, smoking, ethnicity, health insurance, and comorbidities showed that females who consumed ≤2 servings of fruits and vegetables per day were 2.1 times more likely to report poor/fair SRH as compared to those who consumed ≥5 servings per day . The adjusted model explained 11.8% of the variation in SRH . The model explained 12.2% of the variation in SRH . Females who consumed both high and low fat foods were 1.3 times more likely to report poor/fair SRH as compared to those who consumed primarily low fat foods . Logistic regression models adjusted for age, BMI, smoking, ethnicity, health insurance, and comorbidities showed that females who consumed mostly high fat foods were 1.5 times more likely to report poor/fair SRH as compared to those who had primarily low fat foods . Similarly, females who consumed both high and low fat foods about the same were 1.4 times more likely to report poor/fair SRH as compared to those who had primarily low fat foods . The model explained 10.0% of the variation in SRH . The adjusted model showed that males who consumed mostly high fat foods were 3.2 times more likely to report poor/fair SRH health as compared to those who consumed primarily low fat foods . The model explained 14.1% of the variation in SRH . Logistic regression model adjusted for age, BMI, smoking, ethnicity, health insurance, and comorbidities showed that females who reported 1–3 times a month or never of physical activity were 1.6 times more likely to report poor/fair SRH as compared to females who performed ≥5 days per week of physical activity . The model explained 10.3% of the variation in SRH . The association of physical activity and SRH was no longer significant for males once adjustment variables were added   .We found higher odds of poor/fair SRH for males and females who consumed the lowest level of fruits and vegetables. No gender differences were found in a longitudinal cohort of Canadian young adults who were more likely to report excellent SRH if they had a high intake of fruits and vegetables as adolescents . SimilarWe found that both males and females, who consumed mostly high fat foods, were more likely to report poor/fair SRH as compared to those consuming primarily low fat foods. High fat intake was also associated with poorer SRH in African-American adults . ConsistA strong association between physical activity and SRH for females was observed in our study. Females who engaged in less physical activity were more likely to report poor/fair SRH as compared to those who were more physically active. Several studies found physical activity to be related to SRH regardless of gender for a national sample , for a cThe main objective of our study was to assess the relationship of indicators of modifiable cardiovascular disease risk factors as follows: dietary and physical activity lifestyle and their relationship to SRH. We examined SRH in a half-Hispanic population with non-Hispanics who were predominately Black and we found significant differences in ethnicity for SRH. However, it is noteworthy to mention that ethnic differences were adjusted in the final models. Several studies indicated that ethnicity was a factor in the assessment of SRH. A cohort of older Hispanics rated their health poorer than non-Hispanic Whites yet had a lower mortality rate after a 16-year follow-up, based on data from the Health and Retirement Study and adjusting for demographics, socioeconomic status, health status, and health behaviors at baseline . FurtherThere were several strengths of this study. This was a large sample of adults from Miami Dade County, Florida, primarily minorities, half of whom were from a diverse Hispanic population. To our knowledge, this is the first study to investigate the relationship between lifestyle behaviors and SRH for a population largely comprised of Hispanics with a considerable proportion of Blacks. Nevertheless, there are some limitations. Causality of lifestyle behaviors with SRH could not be established due to the single time point. The measurements were self-reported and may be over- or underestimated. Individuals reporting good SRH may have overstated healthy eating and physical activity level. Other factors that may have influenced SRH such as socioeconomic status, neighborhood, housing situation, and psychosocial factors were not collected in this study.Modifiable lifestyle behaviors known to reduce cardiovascular disease risk, low fruits and vegetables intake and high fat intake, were associated with poor/fair SRH in males and females. Poorer SRH was significantly associated with low physical activity in females only, and low physical activity was also more common in women than men in this study sample. These results indicate that gender differences may have implications in designing and monitoring lifestyle interventions to prevent cardiovascular diseases.


Yet, to date, no transcriptome-wide identification of m5C sites has been undertaken in plants. Plants provide a unique comparative system for investigating the origin and evolution of m5C as they contain three different genomes, the nucleus, mitochondria and chloroplast. Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in non-coding ribosomal RNAs and transfer RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba.Post-transcriptional methylation of RNA cytosine residues to 5-methylcytosine  contains supplementary material, which is available to authorized users. In DNA, m5C has been extensively studied due to its ease of detection and functional roles of DNA methylation in eukaryotes have been demonstrated for transcriptional silencing of transposons and transgenes, genomic imprinting and X chromosome inactivation . While iewed in , RNA hasiewed in –4. Transiewed in . Of thesin yeast , 10 and nditions . Furtherit flies . In ribotructure –15.5C methylation is catalysed by two RNA methyltransferases (RMTases); the first class of RMTase is known as tRNA specific methytransferase 4 (TRM4) or NOP2/Sun domain protein 2 (NSUN2), in yeast and animals respectively  supplemented ½ MS media with 1 % sucrose. Hygromycin B was purchased from A.G. Scientific, Inc.. Plants were photographed with a Canon PowerShot G15 camera at 10 and 20 days after germination (DAG).nop2a-2 (oli2-2) (SALK_129648), nop2b-1 (SALK_084427), nop2b-2 (SALK_054685), nop2c-1 (SAIL_1263_B04), nop2c-2 (SALK_149488), nsun5-1 (SALK_204104), nsun5-2 (SALK_004377), trdmt1 (SALK_136635), trm4a (SALK_121111), trm4b-1 (SAIL_318_G04) and trm4b-2 (SAIL_667_D03). The trdmt1 trm4b double mutants were generated using the trdmt1 and the trm4b-1 mutant alleles. The nop2a nop2b double mutants were generated using the nop2a-2 (oli2-2) and the nop2b-1 mutant alleles. Primers used to identify homozygous T-DNA mutants are provided  SALK_29648, noNucleotide sequence data for the following genes are available from The Arabidopsis Information Resource (TAIR) database under the following accession numbers: NOP2A/OLI2 (At5g55920), NOP2B (At4g26600), NOP2C (At1g06560), NSUN5 (At5g26180), TRDMT1 (At5g25480), TRM4A (At4g40000), TRM4B (At2g22400).A. thaliana seedlings, or A. thaliana floral buds, T. durum flag leaf, B. rapa and G. biloba shoot apexes, C. taxifolia fronds or log phase growth N. oculata cells using the Spectrum Plant total RNA kit (SIGMA-ALDRICH) and contaminating DNA removed using DNase (SIGMA-ALDRICH). To enrich for tRNAs, 10μg of total RNA was separated on a 10 % polyacrylamide gel, the region containing 65–95 nts was removed and RNA was purified. Either total RNA or purified tRNAs were used for library construction using Illumina’s TruSeq RNA sample kit v2, as per the manufacturer’s instructions. As bisulfite treated RNA is sheared, bisulfite treated samples were quickly processed after addition of the fragmentation buffer. For bisulfite conversion, 200 pg of control in vitro transcribed Renilla luciferase (R-Luc) RNA was added to either 2 μg of total RNA or 200 ng of purified tRNAs and converted with sodium metabisulfite (SIGMA-ALDRICH) as previously described [Total RNA was isolated from either 10 day old escribed , 51. BisArabidopsis RNA reference sequence for the corresponding rRNA subunit and a consensus was derived in order to obtain species-specific SNPs  was used to align sequence reads to the corresponding tRNA and rRNA reference sequences. In order to compare structural positions in tRNAs with different sequences and lengths, the representative structure model was used [Arabidopsis references and the numbering was based on the nucleotide position of the corresponding Arabidopsis rRNA reference sequence.Sequences were trimmed for adapters and filtered for low quality reads using CLC Genomics Workbench (Qiagen). In order to identify tRNAs and to reduce mapping ambiguity, we collapsed identical and highly similar tRNA isodecoder sequences from The Arabidopsis Information Resource  to creatwas used . For rRN5C. Renilla Luciferase in vitro transcribed mRNA lacking m5C was used to ensure that conversion efficiency was greater than 98 %. To ensure robust detection of methylated sites in tRNAs and rRNAs, a minimum of 5 reads coverage was required and a minimum methylation level of 20 % (≤80 % conversion rate). Percentage methylation at specific positions was calculated as the number of mapped cytosines divided by the combined total number of mapped cytosines and mapped thymines. Heatmaps showing percentage methylation were created in R [In order to identify methylated cytosines, non-conversion of a cytosine in read sequences was taken to indicate the presence of mted in R , using tted in R .Asp(GTC) 72bp PCR product is digested by HpyCH4IV resulting in two digestion products of 35bp and 37bp if C38 is methylated, and is undigested if C38 is non-methylated and thus converted to T38 by bisulfite treatment, causing the HpyCH4IV restriction enzyme site to be lost. The 25S rRNA PCR product is 155bp in length. When C2268 is methylated, the restriction enzyme HinfI cleaves the PCR product into two fragments of 29bp and 126bp. Non-methylated 25S rRNA has a T at position 2268 after bisulfite treatment, and this eliminates the HinfI restriction enzyme site, leaving the product at 155bp and undigested. Undigested PCR products were used as loading controls. Primer sequences used are provided  and helix 70 of 25S nuclear rRNA using the primers forward Asp tRNA_At_FWD and reverse Asp tRNA_At_REV and primers forward 25S_rRNA_F and reverse 25S_rRNA_R, respectively. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four from the 3′ end to generate a HinfI restriction site. PCR products were digested with HpyCH4IV or HinfI restriction enzymes (New England Biolabs), respectively. The tRNATRM4A, TRM4B, NSUN5, NOP2B and NOP2C mRNA was performed using the primers provided (Additional file Semi-quantitative PCR was performed using an Invitrogen SuperScript III kit as per the manufacturer’s recommendations from 2 μg of total RNA and oligo-dT primed cDNA synthesis. Semi-quantitative PCR detection of The data sets supporting the results of this article are available in NCBI’s GEO database repository, and are accessible through GEO Series accession numbers GSE68444, GSE68445, GSE68447 and GSE68448.