The current global conditions, which include intensive globalization, climate changes, and viral evolution among other factors, have led to an increased emergence of viruses and new viral diseases; RNA viruses are key drivers of this evolution. Laboratory networks that are linked to central reference laboratories are required to conduct both active and passive environmental surveillance of this complicated global viral environment. These tasks require a continuous exchange of strains or field samples between different diagnostic laboratories. The shipment of these samples on dry ice represents both a biological hazard and a general health risk. Moreover, the requirement to ship on dry ice could be hampered by high costs, particularly in underdeveloped countries or regions located far from each other. To solve these issues, the shipment of RNA isolated from viral suspensions or directly from field samples could be a useful way to share viral genetic material. However, extracted RNA stored in aqueous solutions, even at −70 °C, is highly prone to degradation. The current study evaluated different RNA storage conditions for safety and feasibility for future use in molecular diagnostics. The in vitro RNA-transcripts obtained from an inactivated highly pathogenic avian influenza (HPAI) H5N1 virus was used as a model. The role of secondary structures in the protection of the RNA was also explored. Of the conditions evaluated, the dry pellet matrix was best able to protect viral RNA under extreme storage conditions. This method is safe, cost-effective and assures the integrity of RNA samples for reliable molecular diagnosis. This study aligns with the globally significant “Global One Health” paradigm, especially with respect to the diagnosis of emerging diseases that require confirmation by reference laboratories. The most recent emerging and re-emerging transboundary animal diseases (TADs) have been an important concern in animal health . ExampleThese examples clearly implicate RNA viruses as key drivers behind the emergence of new variants of existing pathogens. Importantly, RNA viruses have been shown to evolve with the highest mutation rates. Therefore, one of the most important tasks in diagnostic laboratories is to conduct research on the genetic information of these viral agents. This information could be used for viral classification , to tracThe establishment of viral sequence databases has undoubtedly helped to advance the diagnosis and control of these viral diseases. Likewise, several collaborative networks have been created including OFFLU  and the Food and Agriculture Organization of the United Nations (FAO) network of expertise on avian influenza), EPIZONE  and CaribVET . With the goal of integrating the scientific knowledge acquired among the different research laboratories, these networks improve the preparedness, prevention, detection and control of epidemics and contribute to the harmonization and reinforcement of animal disease surveillance.Nevertheless, the exchange of viral isolates or field samples remains the critical point in the collaboration among these different diagnostic laboratories. The virus-containing clinical samples must be stored and transported at temperatures as low as possible, preferably below −70 °C. Storage at this temperature is unreliable . Additionally, some viruses such as classical swine fever virus (CSFV) or foot and mouth disease virus (FMDV) can be transmitted directly by animal contact, and indirectly via contaminated equipment, foodstuff or aerosols (reviewed in ). Hence,To overcome the issues associated with the current methods of shipping unstable and infectious material, a method to effectively transport RNA isolated from viral suspensions or directly extracted from the field samples could be a useful mechanism for sharing viral genetic material between different diagnostic laboratories. However, extracted RNA stored in aqueous solutions, even at −70 °C, is prone to degradation . TherefoInternational standards for animal welfare were used for all animal samples collected, following the regulations of the Institute of Veterinary Medicine (IMV), Ministry of Agriculture (MINAGRI) of the Republic of Cuba. The protocol # PNO-G-040-2016 of the Centro Nacional de Sanidad Agropecuaria (CENSA) was approved by the Committee on the Ethics of the MINAGRI of the Republic of Cuba and all efforts were made to minimize suffering of the animals. The samples were sent directly from the IMV to the Animal Virology Laboratory at the National Center for Animal and Plant Health (CENSA). The IMV is the official regulatory body of the Republic of Cuba; therefore, additional permits were not required.The inactivated isolate A/chicken/Egypt/1709-6/2008 , an HPAI (A/H5N1) virus, was used for all experiments in this study. The virus was re-suspended in 1 mL of sterile phosphate buffered saline (PBS) following the directions of the supplier.Samples of tracheal swabs from chickens free of avian influenza virus (AIV) were collected for use as dilution matrix. The birds sampled were previously tested for AIV following the procedure described by Spackman et al. . The traTotal RNA was obtained from the viral suspension of A/chicken/Egypt/1709-6/2008 inactivated strain and the AIV-free pooled tracheal swabs samples using QIAamp Viral RNA Mini Kit  according to manufacturer’s instructions. In all cases, a starting volume of 150 µL of either tracheal swab samples or the viral suspension was used, and RNA was eluted in 50 µL nuclease free water . The eluted RNA was stored at −80 °C until further use.6 copies/µL, medium RNA load: 104 copies/µL and low RNA load: 102 copies/µL) for in vitro-transcribed RNAs of M and H5 genes. The target regions selected were: (i) the target region reported by Spackman et al. [Different RNA concentrations were fixed  according to the manufacturer’s recommendations. Finally, the RNA transcripts were quantified by spectrophotometry. In this way, a total of 4.2 × 1013 RNA copies/µL for each transcribed gene was obtained. Standard curves based on RNA copy numbers were generated and reaction efficiencies were calculated by the LightCycler software Version 4.05 . The linear ranges for both genes spanned within 107–100 gene copies/µL in terms of RNA copy number.Total RNA obtained from the inactivated H5N1 strain was amplified by RT-PCR using the One Step RT-PCR Kit  following the manufacturer’s directions. The amplified products were checked by 2% agarose gel electrophoresis, stained with ethidium bromide (0.5 g/mL) and purified using QIAquick gel extraction kit . The RT-PCR purified products were directly in vitro transcribed from the T7 polymerase promoter site using a MEGAscript® 2.0  instrument. The quantification of H5 gene was performed using the qRT-PCR assay described in Pérez et al. [In all cases, the synthesis of cDNA was performed by random priming and using M-MLV reverse transcriptase, as previously described in Díaz de Arce et al. . To quanz et al. .2[l2 , 5 µL of template and enough nuclease-free water  for a final volume of 20 µL for each reaction. The quantification of M gene was performed using the published primers targeting the M gene of type A influenza virus, namely forward primer M+25 and reverse primer M-124 , which wFixed RNA loads were stored in different matrices at different temperatures for long-term analysis of RNA degradation. First, the RNA loads were set by spiking in in vitro-transcribed RNA described above into three sets of storage matrices. The different storage matrices consisted of: (i) the pooled elution obtained from samples of tracheal swabs AIV-free (ii) a modified RNA safe buffer (RSB) previously described in Hoffmann et al.  and  to 20 µL of eluted RNA of tracheal swabs from chicken.6, 104, and 102 copies/µL for each gene were finally fixed. To assess the impact of the temperature on the different storage conditions, spiked matrices were stored in parallel at 4 °C, 20 °C and 37 °C, this last condition was considered to simulate a more extreme temperature situation. These three subsets of matrices were analyzed after 3, 7, 10, 14, 21, 27, 41 and 57 days. The RNA was quantified in terms of RNA copy number by qRT-PCR. For intra-assay variability, each dilution was analyzed in triplicate.In vitro-transcribed RNAs were serially diluted 10-fold in each of the three matrices. Three different concentrations  of each in vitro-transcript were used for the experiment. Concentrations of 10The secondary structures of RNA for matrix (M) and hemagglutinin (HA) genes were obtained using the RNAfold program included in the Vienna RNA software package version 2.0.0  . The seqTo assess the stability of M and HA genes under direct action of RNase, the RNA of the inactivated viral strain A/chicken/Egypt/1709-6/2008 was incubated with RNase A dilution . Thus, 150 µL of viral RNA were mixed with 4.5 µL of RNase A dilution (4 mg/mL diluted 1:1000) and incubated at 37 °C for different time periods . The integrity of the diagnostic target regions was assessed by qRT-PCR.® Software 4.05.415 . Significant differences between the means of RNA concentration  stored in each matrix or at each temperature were analyzed by a one-way analysis of variance (ANOVA) using GraphPad Prism version 6.0 software for Windows . A Bartlett’s test was used. A p value < 0.05 was considered statistically significant. The means and standard deviations (SD) of the RNA copy numbers were generated using the LightCycler2, 1.0–1.2 × 104 and 1.2–1.3 × 106 gene copies/µL, for low, medium and high RNA load, respectively, in all three storage matrices  could reflect differences in the methods used to obtain these concentrations. For the fixed RNA loads, the RNA transcripts were quantified by spectrophotometry, whereas, at t = 0 days, the RNA load estimated by was quantified using qRT-PCR, which is based on a fluorometric method. Nevertheless, it is important to highlight that although at t = 0 the RNA load estimated was slightly higher than expected, the magnitude order for the three conditions remained in the desired range.On day 0 (t = 0 days), M gene copies ranged from 2.4–2.8 × 10matrices . The sliThe target region of the M gene at low RNA load was detected by qRT-PCR at each indicated time point in all matrices stored at 4 °C A. HoweveAs with low RNA loads, the target region of the M gene was detected by qRT-PCR in all storage matrices at 4 °C until the end of the experiment when starting with a medium RNA load. The concentrations of the RNA isolated from the swab matrix and the RNA stored in RSB decreased one order (1Log) in comparison with the RNA stored in dry pellet matrix, which was stable until the end of the experiment (57 dpi) B. At a tSimilar trends were observed when starting with high RNA loads. The M gene was detected by qRT-PCR in all storing matrices at 4 °C until the end of the experiment, although the concentrations of the RNA isolated from the swab matrix and the RNA stored in RSB decreased 1Log C. When s2, 1.2–1.3 × 104 and 1.7–1.9 × 106 gene copies/µL, for low, medium and high RNA load, respectively, for all the conditions  was evaluated after RNA storage in different matrices and different temperatures. As for the M gene, at the beginning of the experiment (t = 0 days), the estimated RNA load was slightly higher than expected, ranging from 2.3–2.8 × 10nditions . Howevernditions A. At a tnditions A. At 37 nditions A.For medium RNA loads, the target region of the HA gene was detected by qRT-PCR in all matrices at 4 °C until the end of the experiment B. At a tAt high RNA loads, the target region of the HA gene was detected by qRT-PCR in all three matrices at 4 °C until the end of the experiment. However, the RNA load from the swab matrix decreased one order (1Log), whereas the RNAs stored in RSB and dry pellet matrix were stable until the end of the experiment (57 dpi) C. At a tThe results obtained from the RNAfold program showed that the pattern of conservation of the secondary structure for both HA (H5 subtype) and M genes was highly conserved when strains from the most divergent H5 lineages were compared. In addition, this study also showed that the H5 gene had a higher probability to fold secondary structures when compared to the M gene . The RNAThe stability of the diagnostic target regions for M and HA genes of AIV was assessed by direct action of RNase A and confirmed by quantification of RNA loads . The tarCurrent global factors, which include intensive globalization, climatic changes, and viral evolution, among others, have increased the emergence of viruses and new viral diseases over the last several decades . SignifiThe current study evaluated different RNA storage conditions using RNA transcripts obtained from an inactivated HPAI H5N1 virus as model. The goal of this study was to propose a method to safely transport RNA to different laboratories for reliable downstream applications used in molecular diagnosis. Additionally, this study explore how secondary structures may affect the stability of RNA. The H5N1 virus was selected as model after considering several factors. First, H5N1 virus is an emergent pathogen with an RNA genome for which genetic surveillance programs are necessary in several countries. Therefore, it represents a good example where sample exchange is necessary between different diagnostic laboratories within the global surveillance network . In addi2, 104, 106 copies/µL) based on the fact that they are similar to those found in a natural outbreak caused by influenza viruses [Likewise, the RNA concentrations were selected  are involved in the degradation of RNA in all living organisms . ClinicaResults from this study show that the stability of the M and HA RNA transcripts depended on the temperature, matrix of storage and nature of the gene. Even though at higher RNA loads the target region was detected for most of the conditions established (except 37 °C stored in either swab matrix or RSB), the degradation curves were similar for each starting RNA concentration. Therefore, this result suggests that the RNA degradation rate, in the model studied, was independent of substrate concentration.In terms of optimal storage temperature, the best results of stability over time were obtained at 4 °C, at which all samples tested were detectable at the end of the experiment. This outcome is not surprising given that RNase’s enzymatic activity is known to be lower at this temperature. However, this RNA storage condition can be achieved only by freezing or cooling samples for transportation and comes with all the associated drawbacks related to these methods of preservation as previously discussed (reviewed in ).The only matrix able to protect the RNA from the more extreme conditions  was the dry pellet matrix. Similar results were obtained by Martinez et al. , who werNevertheless, for low concentration of the HA gene incubated at 37 °C, the RNA was detectable only 3 dpi, whereas the M gene, under the same conditions, was detected until the end of the experiment. These results suggest that the nature of each viral gene differentially affects its rate of degradation. Exploring RNA quality to perform gene expression analysis, Opitz et al.  found thThe prediction of secondary structures for the diagnostic target region of the M gene revealed a higher probability of folding compared to the diagnostic target region of the HA gene. In addition, the diagnostic target region of the M gene was more resistant to the RNase A treatment than the diagnostic target region of the HA gene. These results show that secondary structures are important characteristics in the nature of RNA, protecting it from degradation. Therefore, the ability of a target region to fold into secondary structures should be considered for during the diagnostic design. Currently, several thermostable reverse transcriptase enzymes have been developed to prevent failures during reverse transcription reactions caused by secondary structures. In addition, the use of chemical reagent such as 2% DMSO or 2.5 M betaine are known to improve the detection of GC-rich regions . TherefoThe main limitation of our study is that the RNA model used to ensure a reliable quantification of the RNA degradation process was based on RNA in vitro transcripts instead of complete viral genes. Nevertheless, the use of clinical material (elution from negative AIV-free swabs) for the preparation of all matrices assessed, resembles the real environment during sampling process. In addition, the results obtained from this study provide evidence that the RNA degradation process was independent of its length; therefore, similar results could be expected using the complete viral genes. Despite this limitation, our study has contributed to the literature describing a practicable and cost-effective method to exchange samples between different laboratories.In this study, a rigorous assessment of different conditions to store RNA safely and effectively for its downstream applications was performed. Based on our results, the optimal storage matrix is the dry pellet matrix. This storage condition was able to protect the RNA-transcript even under more extreme conditions of storage including higher temperatures and longer periods of time. Therefore, this method is proposed as the method of choice for nucleic acid material exchange between different laboratories. The importance of secondary structures within the diagnostic target regions that prevent degradation of RNA was also highlighted and should be considered in the design of novel target regions. These results point to a method that is safe, cost-effective, and ensures RNA quality for reliable molecular diagnostics. These results have global significance, particularly regarding the development of diagnostic networks for emerging diseases to maintain active and passive environmental and medical surveillance.


The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos  for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development ( Globally, organophosphate (OP) pesticides have been widely used in agriculture, forestry and gardening, which account for almost 40% of total pesticide sales by volume . A BraziS were more neurotoxic in young rats compared with adults [During the past decade, many studies using models based on adult rodents exposed to methamidophos have noted that it affects cardiovascular reflexes, alters spermatogenesis and induces delayed neuropathy ,13,14. Ih adults . In addih adults . Howeverh adults . These ah adults ,23. Furth adults . This mah adults ,26. Thush adults . HoweverThis study was therefore designed to study methamidophos neurotoxicity and related mechanism in the early stages of zebrafish. Two environmentally relevant concentrations of methamidophos (25 and 500 μg/L) were selected, which were according to the concentration in water environment and the level in part of the human samples, respectively ,29. The Methamidophos  was purchased from Dr. Ehrenstorfer . Acridine orange  staining reagent, Hanks’ Balanced Salt Solution  and tricaine  were purchased from Sigma-Aldrich . Total RNA was extracted by TRIzol . Reverse transcription and SYBR-green RT-PCR reagents  were obtained from TaKaRa . All of the other chemicals required in this study were of analytical grade.Danio rerio), acquired from the model animal center of Nanjing University were kept in a recirculating system at 28.5 °C, with a 14 h light/10 h dark cycle 7 days per week [Artemia nauplii, Tianjin Ocean Pal Carol Biotech Co., Tianjin, China) twice a day. Male and female adult fish with a preferable ratio of 2:1 were transferred in pairs overnight in a spawning aquarium, and the spawning finished at the first 30 min of the light cycle on the next morning. Fertilized eggs were collected, washed with embryo medium  and incubated in Petri dishes at 28 ± 1 °C [AB strain zebrafish  and synapsin IIa (syn2a) were analyzed using SYBR Green PCR Master Mix reagent kits . All RT-PCR were performed on an ABI 7900 Fast Real-Time System . The thermal cycle was set at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 1 min. The oligonucleotide primers utilized in this study were synthesized by Invitrogen  and the primer sequences are shown in −ΔΔCt method was used to calculate the relative mRNA expression levels of genes normalized by the β-actin gene [At 72 hpf, after exposure to a series of methamidophos, total RNA was extracted from 20 living zebrafish larvae using Trizol reagent  according to the manufacturer’s instructions, and the concentration of total RNA was measured with NanoDrop 2000 . Synthesis of first-strand cDNA was performed by reverse transcription of 1 mg total RNA with M-MLV RTase . The mRNA levels of tin gene . Each satin gene .p < 0.05. All values were expressed as mean ± standard error of the mean (SEM).The differences between three groups were evaluated by non-parametric test  to identify significant differences. All statistical analysis was conducted using Statistical Analysis System  and the criterion of significance was set at Our experimental flow diagram is presented in In the morphology development analysis, we found the methamidophos-exposed zebrafish larvae did not show obvious delay of morphological development in both 24 hpf and 72 hpf .The number of escape responders for mechanical stimulation was summarized in p < 0.01, respectively for the two exposure groups).We next examined the zebrafish larvae at 72 hpf using AO staining, which could reveal the developmental neurotoxic chemical-induced apoptosis in nervous system. Apoptotic cells of the living larvae in each group were revealed by AO staining. No obvious apoptotic cells were observed in zebrafish larvae of the control group, whereas notable signs of apoptosis appeared in zebrafish larvae of each exposure group, mainly around the brain with bright green fluorescent spots a–c. The mbp, and syn2a genes in zebrafish were detected after methamidophos exposure at 72 hpf  of zebrafish and human [syn2a is a biomarker of synapse formation in mammals, playing an important role in synaptogenesis and neurotransmitter release [mbp expression was significantly down-regulated in larvae from the 25 and 500 μg/L exposure groups, compared with the control    a. Simila groups) b. NotablAs far as we know, this is the first study to report the developmental neurotoxicity and possible mechanisms in terms of exposure to methamidophos in the embryo-larval stages of zebrafish. By the combination of studies regarding larval locomotor behavior, AO staining and transcript expressions, we demonstrated that exposure to environmentally relevant methamidophos in zebrafish embryo and larvae could cause developmental neurotoxicity . Methamidophos is water soluble and has a moderate potential for runoff into surface waters. One of the concentrations of methamidophos exposure (25 μg/L) is close to environmentally relevant concentrations of methamidophos in the water environment of some regions, such as Philippines, India and other countries . BesidesScreening for neurotoxicity can be assessed by motor, sensory, autonomic, and integrative neurological functions. Behavior represents the sum of activities controlled by the nervous system, which can evaluate the consequences of disruption of neuronal communications . BehavioApoptosis, also be referred to as programmed cell death , is an imbp gene was down-regulated in our study, suggesting that exposure to methamidophos might affect the function of oligodendrocytes, and further affect the formation of myelin sheath. Similarly, a recent study shows that exposure to chlorpyrifos, which is another widely used OP, resulted in reduced mbp in the early life stage of zebrafish [syn2a was increasingly expressed during the process of nervous system differentiation, implying that synapsins is crucial for neuronal differentiation and synaptogenesis in the early developmental stages of CNS [syn2a observed in this study may affect neuronal differentiation and synaptogenesis, and ultimately lead to neurobehavioral impairments.It is widely demonstrated that the structure, synthesis and gene expression of myelin sheath are highly conserved between zebrafish and mammals ,62. The ebrafish . Recentls of CNS . TherefoThe developmental neurotoxicity of methamidophos has been studied in rodents. Moser found that methamidophos  could alter neurobehavior in young rats . Lima etThis is the first study to reveal the induction of developmental neurotoxicity in zebrafish by environmentally relevant concentration of methamidophos. Methamidophos decreases the number of escape responders for mechanical stimulation through the action of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain in embryo-larval stages of zebrafish. Our study provides novel insights into methamidophos induced developmental neurotoxicity and its underlying mechanism, and highlights the importance on studying the developmental neurotoxicity of methamidophos in other aquatic species and humans.


Objectives: The main goal of this study was to investigate and compare the neural substrate of two children's profiles of high intelligence quotient (HIQ).Methods: Two groups of HIQ children were included with either a homogeneous (Hom-HIQ: n = 20) or a heterogeneous IQ profile (Het-HIQ: n = 24) as defined by a significant difference between verbal comprehension index and perceptual reasoning index. Diffusion tensor imaging was used to assess white matter (WM) microstructure while tract-based spatial statistics (TBSS) analysis was performed to detect and localize WM regional differences in fractional anisotropy (FA), mean diffusivity, axial (AD), and radial diffusivities. Quantitative measurements were performed on 48 regions and 21 fiber-bundles of WM.Results: Hom-HIQ children presented higher FA than Het-HIQ children in widespread WM regions including central structures, and associative intra-hemispheric WM fasciculi. AD was also greater in numerous WM regions of Total-HIQ, Hom-HIQ, and Het-HIQ groups when compared to the Control group. Hom-HIQ and Het-HIQ groups also differed by their hemispheric lateralization in AD differences compared to Controls. Het-HIQ and Hom-HIQ groups showed a lateralization ratio (left/right) of 1.38 and 0.78, respectively.Conclusions: These findings suggest that both inter- and intra-hemispheric WM integrity are enhanced in HIQ children and that neural substrate differs between Hom-HIQ and Het-HIQ. The left hemispheric lateralization of Het-HIQ children is concordant with their higher verbal index while the relative right hemispheric lateralization of Hom-HIQ children is concordant with their global brain processing and adaptation capacities as evidenced by their homogeneous IQ. Children with very high intelligence quotient (HIQ)  to calculate the FSIQ. Children scoring 130 or greater in VCI or PRI were included in HIQ groups. Children with a VCI significantly higher than the PRI were included in the Het-HIQ group, while those with no significant difference between VCI and PRI were included in the Hom-HIQ group. The significance of VCI-PRI differences was defined by the WISC-IV score with a range of 15–46 in the Het-HIQ group. Since the number of Het-HIQ children with significantly higher PRI than VCI was under represented (3 subjects out of 27), these subjects were excluded for group homogeneity purposes.A total of 44 right-handed  for children from ages 4 to 16  .MRI examinations were performed without any sedation at the MRI department of CERMEP-Imagerie du Vivant, on a 1.5T Siemens Sonata system  with an 8-channel head-coil and 40 mT/m gradients. The MRI protocol included a 3D T1-weighted magnetization prepared rapid gradient echo (MPRAGE) sequence  were applied with a nominal isotropic resolution of 2.5 mm3. The b0 image was acquired four times to increase signal to noise ratio while the other directions were acquired twice. This protocol follows international recommendations on DTI acquisition  sequence . Fifty-one contiguous axial slices, of 2.5 mm thickness were acquired in the anterior commissure—posterior commissure (AC-PC) plane. Twenty-four diffusion gradient directions  in order to detect artifacts arising from subject motion or scanner malfunction, and confirm the lack of visually detectable abnormalities.Analysis of diffusion tensor data was performed using the Functional Magnetic Resonance Imaging of the Brain (FMRIB) Software Library (FSL) . The anatomical location of significant clusters was identified based on WM atlases (JHU ICBM-DTI81 White Mater Labels and JHU White-Matter Tractography Atlas) in FSL. In order to confirm the voxel-wise analysis results, quantitative values of diffusion metrics were extracted from 48 regions of interest (ROI) and 21 fiber-bundles of the JHU atlases, by multiplying atlas labels with the TBSS skeleton obtained from all 57 subjects.The employed voxel-wise analysis was based on a non-parametric approach using the permutation test theory with a standard generalized linear model design matrix. By allowing inference on the statistical maps when the null distribution is not known, this approach provides an easily implementable solution to the multiple testing problems. The permutation testing was performed using the Randomise module of FSL. Group comparisons were performed between each group of HIQ children and control subjects with age and gender as co-factors. The threshold-free cluster enhancement (TFCE) option of Randomise was applied on the resulting statistical maps. This method enhances cluster-like structures in an image without having to define an initial cluster-forming threshold or to carry out a large amount of data smoothing. The resulting statistical parameter maps were corrected for sex and age and for multiple comparisons by the family-wise error rate (FEW-corrected https://www.r-project.org). ANOVA followed by Tukey's post-hoc tests were performed on demographic data, IQ, CBCL, and CPRS scores, and DTI metrics (ROI and fiber bundles) to detect group-wise differences between the four children groups  with age, gender and CBCL as co-factors.Statistical analysis was performed using the R library (p = 0.07) and thought (p = 0.03) problems, as reported in Table Descriptive statistics of age and IQ scores are reported in Table Figures When compared to the Control group, TBSS analysis showed significant higher AD values in Total-HIQ, Hom-HIQ, and Het-HIQ groups as illustrated in Figure When comparing the localization of these AD increases, we observed a significant difference in hemispheric lateralization. As illustrated in Figure TBSS analysis showed significant higher FA values in Hom-HIQ compared to Het-HIQ groups Figure . FA diffBased on the JHU atlases, the diffusion metrics were measured in different ROIs (ICBM DTI-81) and fiber-bundles (White-Matter Tractography) in HIQ and Control groups to confirm our voxel-wise findings. When comparing Hom-HIQ and Het-HIQ groups, FA values were significantly increased in several ROIs, the body of corpus callosum, the left inferior cerebellar peduncle (ICP), the left posterior corona radiata (PCR), and the right fornix stria terminalis (FX/ST), and in one fiber bundle, the cingulum cingulate gyrus (CCG). When comparing with the Control group, AD values were significantly increased in 17 ROIs and six fiber bundles of the Tot-HIQ group, in four ROIs and three fiber bundles of the Hom-HIQ group, and four ROIs and two fiber bundles of the Het-HIQ group, as reported in Table Effects of full-scale IQ, and CBCL score (Achenbach) on AD values were estimated using General linear models for WM region reported in Table In this work, TBSS analysis was performed to investigate the diffusion-related differences in HIQ children, and between two groups of HIQ, based on their IQ profile, namely homogeneous and heterogeneous. To our knowledge, this work is the first to investigate WM microarchitecture in these two profiles of HIQ children.When compared to the Control group, both HIQ groups presented greater AD and FA in widespread WM regions of frontal, central and associative pathways. Central regions included mainly the body and the genu of the corpus callosum, the fornix and the cingulum as well as brainstem and cerebellar regions. These findings are in agreement with previous DTI studies on intelligence showing a bilateral fronto-parietal network as a neural substrate of enhanced information processing and intelligence  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the CPP Sud-Est IV (France).FN coordinated the organization and implementation of the study, recruited the children, provided the clinical expertise in the interpretation of the results, and participated to the paper writing. SH performed the voxelwise analysis and participated to the statistical analysis, and to the paper writing. GK performed the regional analysis and participated to the statistical analysis, and to the paper writing. CS helped in the interpretation of the results and participated to the paper writing. PF participated to the coordination of the study. OR participated to the coordination of the study, recruited children and provided the clinical expertise in the interpretation of the results. DSM supervised MR acquisition, provided methodological expertise in the interpretation of the results, and participated to the paper writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A‐modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady‐state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady‐state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2‐depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging.Gene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post‐transcription, and post‐translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6‐methyladenosine (m6A)  Recent studies using high‐throughput RNA sequencing technology have revealed that the expression of mammalian mRNAs changes extensively during the aging process  capping, which can regulate many cellular processes by influencing RNA metabolism , N6‐methyl adenosine (m6A), N1‐methyl adenosine (m1A), 5‐methylcytosine (m5C), and 2‐O‐methylation (2'OME). Additionally, di‐methylation occurs when a combination of two different methylations affect the same mRNA, such as when m6Am is observed next to the 5' cap  m6A‐modified RNA collected from 11 young and 11 old individuals  fragments in the young PBMCs and 826 in the old PBMCs with 778 overlapping sites Figure . m6A RNANext, we investigated whether m6A modification affects mRNA abundance in general using a young versus old cohort comparison. We first compared the relationship between an RNA abundance and degree of m6A methylation by plotting FPKM of m6A‐modified RNA fragments on the X‐axis and total RNA on the Y‐axis within the same cohort group Figure a,b. Our We also analyzed m6A‐modified RNA sequences adjacent to the translation start and stop codons of mRNA, and then identified that the preferential region of m6A modification on mRNAs is similar to each other Figure . This ob2.2DROSHA mRNA was highly methylated both in young and old PBMCs with no significant change, while methylation of AGO2 mRNA decreased in the old PBMCs . The total RNA transcriptome of PBMCs revealed that AGO2 mRNA is underexpressed in old PBMCs while the levels of DROSHA mRNA did not change significantly . We observed a decline of m6A modification in late passage IMR‐90 (PDL‐42) similar to observations in human PBMCs   of AGO2 mRNA stability from 4.9 hours to 9.5 hours and 7.1 hours, respectively, but not DROSHA, DICER1, and GAPDH mRNA did not show a drastic change, which supports our observation  (Data not shown). This finding suggests that m6A modification on AGO2 mRNA stabilizes it in HDFs. A possible explanation is that not all m6A methylation elicits the same consequence on mRNA stability.The possibility of m6A‐mediated regulation of 2.4AGO2 mRNA in old PBMCs implicate a change in miRNA abundance as observed in mouse embryonic fibroblasts lacking AGO2  miRNA abundance is regulated by AGO2 mRNA stability modulation, 2) the stabilization of AGO2 mRNA is methylation‐dependent, and 3) AGO2 mRNA decay contributes to senescence and aging.Our study reveals an m6A methylation profile of human aging, regulation of If the expression levels of DROSHA, DGCR8, and DICER1 are crucial for miRNA abundance in cellular senescence and aging, then we should observe that almost all miRNAs are underexpressed due to the depletion of DROSHA, DGCR8, and DICER1 in senescent HDFs and old PBMCs. However, senescent HDFs exhibit a subset of miRNAs that are either overexpressed or underexpressed relative to proliferating HDFs   and old (64 years) participants from the “Healthy Aging in Neighborhoods of Diversity across the Life Span study” (HANDLS) , . Transcript levels were normalized to the abundance of 18S rRNA.Human IMR‐90 cells (PDL‐12) were cultured in DMEM (Invitrogen) supplemented with 10% (v/v) FBS and antibiotics. Plasmids were transfected at 1–2 μg/ml . Transfected cells were analyzed 48 h later after actinomycin D treatment (2.5 μg/ml) at different time periods as indicated. At each time point, total RNA was isolated using TRIzol according to the manufacturer's instructions, and used for RT‐qPCR analysis to determine RNA half‐life . qPCR was carried out using SYBR green master mix (Kapa Biosystems), and a thermal cycler (Bio‐Rad). The primers used for RT‐qPCR are provided in Table 4.4p value <.05, absolute value of Z ratio >1.5, and with FDR <0.3 for significant change.We analyzed global m6A modification, transcriptome, and microRNA expression from human participants as follows. We first isolated total RNA from the human study participants using TRIzol (Invitrogen). For m6A profiling, 1 ug of total RNA was fragmented using Bioruptor for 30 min, resulting in 100–200 nt RNA fragments. Immunoprecipitation of small RNAs with an m6A‐specific antibody (from Synaptic Systems) enriched the amount of methylated RNAs in the immunopellets. Once m6A RNAs are recovered, small RNA library preparation with the Illumina small RNA library preparation kit (Illumina GAII). The libraries were run in GAII, and the resulting data were aligned to human genome sequences (HG19) for quantitation and motif determination. For transcriptome analysis, we followed the standard protocol of Ion Torrent Proton sequencing. For microRNA profiling, Agilent human miRNA Microarray 15.0. was performed and individual miRNAs with pairwise z‐test 4.5Whole‐cell lysates, prepared in radioimmunoprecipitation assay (RIPA) buffer, were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto nitrocellulose membranes (Invitrogen iBlot Stack). Primary antibodies recognizing DROSHA, DICER1, METTL3, and Actin were purchased from Cell Signaling Technology. AGO2 antibody was purchased from Abcam. Horse Radish Peroxidase (HRP)‐conjugated secondary antibodies were purchased from GE Healthcare.4.6◦C. After the beads were washed with NT2 buffer , bead‐associated RNA was extracted with acidic phenol. Isolated RNA was further assessed by reverse transcription (RT)‐quantitative polymerase chain reaction (qPCR) analysis using AGO2 mRNA primers  for 3 hr. The complexes were incubated with protein A‐sepharose beads for 2 hr at 4rs Table . 18S rRNrs Table  were use4.72 fold changes expression was performed in R. For each group of interest, the empirical cumulative distribution functions of log2 fold mRNA expression values were computed using the ecdf function, and pairs of curves to be compared were displayed with plot.cdf. Significance of difference between the cumulative distribution curves was determined using the KS (Kolmogorov–Smirnov) test, and the corresponding p‐value was included in the plot.Cumulative distribution analysis of mRNA log Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Twenty-five genes, including LRP11 and PLIN5, had editing sites that were associated with plasma lipid levels. Our findings provide new insights into the genetic regulation of RNA editing and establish a rich catalogue for further exploration of this process.RNA editing modifies transcripts and may alter their regulation or function. In humans, the most common modification is adenosine to inosine (A-to-I). We examined the global characteristics of RNA editing in 4,301 human tissue samples. More than 1.6 million A-to-I edits were identified in 62% of all protein-coding transcripts. mRNA recoding was extremely rare; only 11 novel recoding sites were uncovered. Thirty single nucleotide polymorphisms from genome-wide association studies were associated with RNA editing; one that influences type 2 diabetes ( Alu repetitive elements. These short interspersed nuclear elements are ∼300 base pairs (bp) in length, specific to primates, and often embedded in introns and 3′ untranslated regions (UTRs). The abundance of Alus in the human genome—more than one million copies—makes it very likely to encounter inverted pairs of Alus, which can form stem-loop structures that are favored substrates of ADAR. Cytosine-to-uracil (C-to-U) editing is much less frequent than A-to-I editing  will capture the edited base . A discrStockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET)   study. WFirst, we focused on patterns of global RNA editing. To identify clinical features associated with RNA editing, we mapped RNA editing quantitative trait loci (edQTLs). Finally, to assess the disease relevance of RNA editing, we analyzed our edQTLs in the context of published genome-wide association studies (GWAS).P-values were calculated with Welch’s t-test. All statistical tests were two-sided.R v.3.2.2 was used for all statistical analyses . If not n = 538) and internal mammary artery (n = 552) were exclusively prepared from rRNA-depleted libraries generated with the Illumina RiboZero protocol. The remaining samples were almost all poly(A)-enriched. Of the 4301 samples included in this study, 52%  were sequenced with a strand-specific protocol, and the remaining were sequenced with a non-strand-specific protocol. A complete summary of tissues, samples and protocols is shown in RNA-seq and genotype data (generated with the Illumina Infinium OmniExpressExome-8 chip) in this study originated from samples collected of up to 7 tissues  and 2 cell types (macrophages and foam cells)  from 855Metabolite and protein data were generated by Olink AB (Sweden) and Nightingale .https://github.com/oscar-franzen/rnaed.The human genome GRCh38 and GENCODE  v.24 annSequencing reads were aligned with the human genome using STAR  v.2.5.1b121.2 billion single-end reads were mapped to unique locations in the human genome . Collapsing PCR duplicates reduced the numbers to ∼42.9 billion aligned reads . In total, 22,124,713 RNA editing events were called, mapping to 2,631,392 unique sites. 83.3%  and 92.6%  of the identified events were A-to-G mismatches in strand-specific and non-strand-specific libraries, respectively. In the non-strand-specific data, these numbers also include the reverse complement of A-to-G .Sequencing errors are more likely to occur toward the end of reads. We did not find 5′ or 3′ positional biases in sequencing reads for canonical events . Therefore, we assessed the reproducibility of RNA editing in 235 macrophage—foam cell pairs. We first assessed the percentage of editing sites detected in both samples in each pair; the median across all pairs was 24%. It may therefore be plausible that many RNA editing sites are not strictly reproducible even in samples that are close to biological replicates. This may reflect the stochastic nature of RNA editing or variability in sequencing depth. We also determined whether the editing ratios were stable for the ∼24% of sites found in both samples of each pair. Across each of the 235 macrophage-foam cell pairs, the median Spearman correlation was 0.83 . Thus, dGene expression was calculated as RPKM  from rearntransform in the R package GenABEL ; (ii) R-sq > 0.4; and (iii) no correlation between gene expression (of the overlapping gene) and the tested clinical parameter .Association-testing was conducted with the linear model in MatrixEQTL  v.2.1.1. GenABEL  with tiep-values, which were used to adjust the real p-values. An adjusted p-value <0.20 was considered significant.Genotypic data were processed as described ; biallelP-values were adjusted with the Benjamini–Hochberg procedure and adjusted P < 0.05 was considered significant. For each editing site, all overlapping genes were tested.Normalized gene expression data from  were useP < 1e − 08. GWAS SNPs that were also edSNPs were saved in a new list that was LD-pruned with PLINK (–indep-pairwise 400 kb 1 0.6)  .n = 538; non-atherosclerotic arterial wall , n = 552; liver, n = 545; skeletal muscle, n = 533; visceral abdominal fat, n = 533; subcutaneous fat, n = 532; whole blood, n = 559; macrophages, n = 256; and foam cells, n = 235). In addition, 18 coronary artery samples were included. Sequencing was done by using 50-bp single-end reads  and 100-bp single-end reads . Differences in read-length and strand specificity affected the sensitivity to detect RNA editing, and most analyses were therefore done separately on these two sets of samples. See To characterize the human RNA “editome”, we analyzed RNA-seq data from the STARNET  study, in = 117, 493). A-to-G(I) editing was detected in transcripts from 25,951 genes, of which 62.1%  were protein-coding. We intersected RNA editing events with genome annotations . T-to-C events have been reported in studies utilizing high-throughput sequencing data but it is unknown whether T-to-C events are authentic or artifacts . Interestingly, in strand-specific libraries the number of A-to-G(I) events correlated with the number of T-to-C events , suggesting that detection of RNA editing per sample was not saturated or that the mechanisms mediating RNA editing are nonspecific. If RNA editing is nonspecific, it can be assumed that deeper sequencing does not saturate detection. Analysis of the cumulative number of unique A-to-G(I) sites as a function of added samples did not reveal a clear tendency toward saturation . Alu was the sole element significantly enriched in editing events  had at least one A-to-G(I) editing event. Despite covering 20.7%  of the genome (Alu A-to-G(I) compared to editing sites in Alus , likely reflecting the strong preference of ADAR for double-stranded folds. When Alu elements were grouped by subfamilies  according to their established phylogeny  and AluY (4.5%), see AluY , perhaps because purifying selection lowered editing levels in mRNAs.In the present version of the human genome assembly (GRCh38) there are 1,186,920 g events –S9. Overe genome , LINE elhylogeny , it showADAR1, ADAR2, and ADAR3 was compared across the tissues/cell types ; the lowest levels observed were in skeletal muscle , see ADAR1 correlated with A-to-G(I) editing events in every tissue except foam cells , further corroborating the authenticity of called RNA editing events. Whole blood showed the strongest correlation between A-to-G(I) editing events and ADAR1 expression , liver (P = 0.049), and subcutaneous fat  events only in atherosclerotic aortic wall  and was stronger in females . However, there were no sex-specific differences in editing in any tissues (P > 0.05). ADAR3 was essentially below the detection level in all tissues .RNA editing provides a mechanism for cells to generate additional protein diversity . HoweverEditing ratios had a long-tailed skewed distribution (median = 0.14), suggesting that these variants are present in a small number of transcripts. Most of the non-synonymous sites  were found in single samples. Thus, although variants are pervasive on a global scale, many reflect background noise.conserved = 0.30, mediannotconserved = 0.05, P < 2.2e−16, Mann–Whitney U test). Moreover, 80% (52/65) of these sites had been reported in REDIportal (GRIA2 (glutamate ionotropic receptor AMPA type subunit 2) was found at the known site    in aortiEleven recoding sites were novel . Seven hIGFBP7 (insulin-like growth factor-binding protein 7). The first site, chr4:57,110,062 changes a codon encoding lysine to arginine (amino acid 97); it was found specific to the internal mammary artery and had been reported in brain  were previously reported only in mouse .PDZD7 (PDZ domain containing 7). It was specific to visceral abdominal fat and modifies the stop codon UAG to UGG (encodes tryptophan), extending PDZD7 by 18 amino acids (WSQIPGLKLSTRLSLPKC). This recoding site was recently reported in human brain tissue . Using the same criteria described above, we found one recoding site, at chr10:101,017,585 in n tissue . To our P = 2.2e−05; FDR = 8.5%) and chr10:51,299,636 .In preparing the sequencing libraries, we used the Ribo-Zero protocol to eliminate ribosomal RNA, which enabled us to investigate small non-coding RNAs in 996 RNA-seq samples from atherosclerotic aortic wall and internal mammary artery. We identified A-to-G(I) editing sites in 102 unique microRNAs  and 73 small nucleolar RNAs (snoRNAs). Stringent editing criteria (recurrence in > 50 samples) identified 18 editing sites in 10 miRNAs  and PLIN5 (lipid storage droplet protein 5). In subcutaneous fat, the LRP11 editing site , located in the most 3′ intron of the longer isoform, was associated with plasma low density lipoprotein (LDL) levels. RNA editing of two genes known for their roles in innate immune response—C3 (complement component 3) and C1RL (complement C1r subcomponent like)—occurred in liver and were both associated with plasma HDL levels.Although none of the 25 genes had been directly associated with coronary artery disease, 22 of 25 sites were associated with plasma lipoprotein levels. Two of the affected genes may have roles in lipid metabolism: trans (the regulatory SNP and the editing site are located on different chromosomes).To uncover possible genetic regulation of RNA editing , we lookAlu elements ; the Alu-subtype distribution was the same as shown in Most of the editing sites with edQTLs were enriched in rs2573346 is associated with sarcoidosis . rs2028299 is associated with expression of ARPIN in fat tissue (P < 1e−10) . rs78579285 has been associated with joint mobility (Beighton score) (PIEZO1 (encodes a mechanosensitive ion-channel component). rs7546668 is associated with kidney function (glomerular filtration rate) (DNAJC16 (encodes DnaJ Heat Shock Protein Family (Hsp40) Member C16) and AGMAT (encodes Agmatinase). rs4073054 is associated with HDL  and HDL . rs1127311 is a pleiotropic locus and it is associated with C-reactive protein and triglyceride levels , which is associated with Parkinson’s disease . In liver, there was a negative correlation between expression of APOC1P1 and APOE  as well as APOC1P1 and APOC1 . The negative correlation may indicate a regulatory function of APOC1P1. rs1883350 is associated with fatty liver disease (PNPLA3 (Patatin-like phospholipase domain-containing protein 3). PNPLA3 is associated with fatty liver disease . The editing sites are located 64 bp apart and are intriguing since they have opposite effect sizes. TTPA is associated with the severity of atherosclerotic lesions in the proximal aorta  . A negatn score)  and we don rate)  and it fon rate) . The ediwith HDL  and had e levels . Interesn levels . rs3947  disease . rs49570 colitis , and it SLC22A25 . rs67565et count  and it h disease  and had ts locus  has been disease  and we i disease  and encofarction  and we fal aorta .We analyzed RNA editing by revisiting RNA-seq data from the STARNET study . The stuAlu elements, as previously reported  three letter tissue abbreviation; (2) library type ; (3) chromosome; (4) position; (5) DNA base; (6) RNA base; and (7) number of samples in this tissue-library combination where this event was detected. Coordinates are in GRCh38.Click here for additional data file.10.7717/peerj.4466/supp-4Dataset S2p-value of the association between RNA editing and the regulatory SNP; (8) adjusted (permutation-based) p-value; (9) the gene overlapping the RNA editing site; (10) same as previous, but giving the gene symbol instead; (11) gene biotype according to GENCODE; (12) the repeat type, if any, overlapping the RNA editing site; (13) the trait, if this regulatory SNP is a reported GWAS lead SNP; (14) gene region; (15) if the gene has previously reported as involved in cardiometabolic traits; (16) if the RNA editing site has been reported in DARNED; (17) if the RNA editing site has been reported in REDIportal; and (18) if the overlapping gene has an eQTL. Abbreviations: (Y)es, (N)o. This table is given as an external file.Supplementary material, RNA editing in metabolic and vascular human tissues The table is tab separated. The columns correspond to: (1) three letter tissue abbreviation; (2) regulatory SNP; (3) the encoded (effect) allele of the regulatory SNP; (4) genomic coordinate of the RNA editing site (GRCh38); (5) molecular interaction type ; (6) beta coefficient; (7) Click here for additional data file.


We show that the affinity to NAD+ and UDP-containing factors during initiation is much lower than their cellular concentrations, and that initiation with them stimulates promoter escape. Efficiency of initiation with NAD+, but not with UDP-containing factors, is affected by amino acids of the Rifampicin-binding pocket, suggesting altered RNA capping in Rifampicin-resistant strains. However, relative affinity to NAD+ does not depend on the −1 base of the template strand, as was suggested earlier. We show that incorporation of mature cell wall precursor, UDP-MurNAc-pentapeptide, is inhibited by region 3.2 of σ subunit, possibly preventing targeting of RNA to the membrane. Overall, our in vitro results propose a wide repertoire of potential bacterial RNA capping molecules, and provide mechanistic insights into their incorporation.Bacterial RNA polymerase is able to initiate transcription with adenosine-containing cofactor NAD+, which was proposed to result in a portion of cellular RNAs being ‘capped’ at the 5′ end with NAD+, reminiscent of eukaryotic cap. Here we show that, apart from NAD+, another adenosine-containing cofactor FAD and highly abundant uridine-containing cell wall precursors, UDP-Glucose and UDP-N-acetylglucosamine are efficiently used to initiate transcription  Escherichia coli was known to be able to start RNA synthesis with cellular nucleotide coenzymes, adenosine derivatives NAD+ (nicotinamide adenine dinucleotide), NADH (reduced form of NAD+) and FAD (flavin adenine dinucleotide)  initiated with adenosine triphosphate (ATP), NAD+ and dpCoA were solved for Thermus thermophilus RNAP (For few decades multi-subunit RNA polymerase (RNAP) from leotide) . Authorse 5′ end . In 2015quencing . It traninvolved . Correlalus RNAP  demonstrEukaryotic mRNA turnover depends on the efficiency of cap removal. Major catalytic role in decapping in eukaryotes is played by NUDIX motif-containing protein Dcp2p. In bacteria, NudC (NADH pyrophosphohydrolase), which contains NUDIX motif was shown to de-cap RNAs from NAD+ . Notablyet al. reported 3- to 4-fold increase of in NADylated RNAI stability in ΔNudC cells (lacking de-capping activity)  and its possible role in transcription. We also show that the repertoire of potential capping molecules is wider, and includes uridine containing precursors of cell wall synthesis (UDP-glucose and UDP-N-acetylglucosamine). Our data also suggest the role of region 3.2 of initiation factor sigma in guarding transcription against incorporation of ‘long-tailed’ NTP analogues into RNA.ATP and UTP were from GE Healthcare; AMP, ADP, NAD+, NADH, NADP+, FAD, UMP, UDP, UDG-Glc and UDP-GlcNAc were from Sigma Aldrich; UDP-MurNAc pentapeptide was a kind gift from Prof. Vollmer.E. coli rpoB gene were constructed by site-directed mutagenesis in polycistronic expression plasmid pGEMABC, coding for RNAP core subunits α, β and β’ (E. coli T7 express strain (New England Biolabs) together with ω subunits from expression plasmid pRSFD_2_rpoZ  or σS and 2 pmols of promoter-containing linear DNA fragment were incubated at 37°C for 10 min in 10 μl of transcription buffer (20 mM Tris–HCl (pH 7.9), 40 mM KCl, 0.1mM ethylenediaminetetraacetic acid) at 37°C, then nucleotides or nucleotides analogues were added to the final concentration of 500 μM (unless otherwise indicated). Transcription was initiated by the addition of 10 mM MgCl2, 50 μM (α32P)-CTP, 12.5Ci/mmol  for RNAI and acnA templates; for T7A1 template 50 μM (α32P)-UTP, 12.5 Ci/mmol of were used. Reactions were stopped after 3 min incubation at 37°C (unless otherwise indicated) by the addition of formamide-containing loading buffer. For the kinetics of synthesis of 9 nt-long transcript from RNAImod, 500 μM ATP or NAD+ were incubated with promoter complex and then 20 μM UTP and 20 μM (α32P)-CTP, 12.5 Ci/mmol were added in the absence or presence of 5 μg/ml of Rifampicin. Reactions were stopped after periods of time indicated on Figure 32P)-CTP, 12.5 Ci/mmol, were added and reactions were incubated for 10 min. For similar experiment on Figure 32P)-CTP, 12.5 Ci/mmol, were added and reactions were incubated for 10 min. For apparent Km determination on RNAImod and acnA templates NTPs and analogues were used in concentrations ranging from 10 μM to 10 mM and constant 50 μM CTP (second NTP for both templates) concentration. Reactions were incubated for periods of time chosen to get approximately the same intensities of the product bands. The bands intensities were quantified using ImageQuant software; to calculate the initial reaction rate these numbers were divided by reaction duration time. These data were fitted to hyperbolic equation using non-linear regression procedure in SigmaPlot software.A total of 0.3 pmols of wild-type or mutant RNAP core with 1 pmols of σE. coli RNAP σ70 holoenzyme on a linear DNA containing promoter RNAI (that was shown to be the most heavily modified by NAD+ in E. coli). ATP or co-enzymes (each at 500 μM concentration) -CMP (at 50 μM). Position +3 of the RNAI promoter was mutated to T to make RNAImod template (here and after we refer to the sequence of the non-template strand) to preclude extension of RNA further than position +2, which would obscure kinetics analysis -UMP on a strong promoter A1 from bacteriophage T7, where transcription also starts with A .Abortive production of short oligonucleotides is the very first stage of the synthesis of the RNA. These products can be resolved in high density denaturing polyacrylamide gel. We analysed the efficiency of incorporation of potential cap molecules into RNA in abortive synthesis by s Figure . As can s Figure  and the s Figure . EfficieA Figure . NADP+ wA Figure . TheKms In the presence of CTP and UTP, the short transcripts initiated with NAD+, NADH and FAD on the RNAImod template were efficiently extended into 9-nt products Figure . The amoKm for NAD+ and ATP on these four promoters variants in abortive synthesis as above .Nicotinamide moiety may, in theory, form base pair with template DNA base at the position −1. This may change specificity of NADylation at promoters with particular base at −1 position. It was observed that A to C (non-template strand) change in −1 position decreased the overall efficiency of NADylated transcript synthesis . To teste Figure . We founting ATP . Therefoting ATP . Note thWe tested if there are any other structural determinants for cofactors incorporation apart from −1 position of template. 5′ NTP of the growing transcript passes through the Rifampicin-binding pocket of the β subunit of RNAP. It was shown that γ-phosphate of the initiating ATP is in proximity to the β subunit fragment between amino acids 516 and 540 , suggestS, while housekeeping σ70 operates in exponential phase. We analysed if different initiation factors may dictate specificity towards capping cofactors. To answer this question we tested initiation with NAD+ on RNAI promoter with holo σ70 and holo σS  The results suggest that at least the two initiation factors tested do not provide significant specificity towards utilization of NAD+ as initiation substrate.A larger proportion of RNAI was found to be NADylated in stationary growth phase compared to the exponential phase . In statS Figure  and D. TS Figure  and D. K70 or mutant σ70 lacking region 3.2, σ70Δ3.2  on RNAImod template. A relatively low concentration of CTP and UTP (20 μM) allowed us to monitor the accumulation of short RNAs ranging from 2 to 9 nt in length. As can be seen from Figure E. coli cells contain high concentrations of a number of nucleotide analogues potentially capable of initiating transcription. One of the most abundant small molecules in E. coli cell, second only to ATP, is UDP-N-acetylglucosamine (UDP-GlcNAc)  , which p-GlcNAc)  can init-GlcNAc) , the las32P)-CMP (at 50 μM concentration). As can be seen from Figure E. coli RNAP can efficiently incorporate UDP-Glc and UDP-GlcNAc . TheKm values on the acnA promoter for UDP-Glc and UDP-GlcNAc were 300 ± 62 μM and 333 ± 41 μM, respectively (compared to 120 ± 17 μM for UTP). These Km values are well below intracellular concentrations of UDP-Glc and UDP-GlcNAc in exponential growth phase . We analysed if dinucleotides initiated with UDP-Glc and UDP-GlcNAc can be extended to facilitate escape into elongation. We mutated acnA promoter  by holoenzymes formed with wild-type σ70 or σ70Δ3.2. As can be seen from Figure Interestingly, UDP-MurNAc pentapeptide was not utilized by RNAP at all. It is possible that RNAP possesses a mechanism that prevents incorporation of UDP-MurNAc pentapeptide into an RNA, which would be too costly for cells and may lead to unwanted targeting of the modified transcript towards the membranes. One of the possible obstacles for the pentapeptide in the active site could be region 3.2 of σTo test if Rif-pocket plays role in selectivity of UDP-GlcNAc utilization in initiation, as it does for ADP-containing co-factors, we tested the activities of Rifampicin resistant mutant RNAPs that we used above. Mutant RNAP with Q513I substitution was inactive on acnA even with UTP, and was excluded from analysis. The rest of mutations in Rif-pocket did not affect incorporation of UDP-GlcNAc into the transcript strongly Figure , suggestin vivo, another adenine containing co-factor FAD can also be incorporated by RNAP in sequence-dependent manner, and thus cap RNAs on +1A (T in the +1 position of the template strand) promoters. Furthermore, we show that UDP-containing cell wall precursors may efficiently cap RNAs on +1U promoters.Here we showed that RNAP is able to incorporate variety of cellular cofactors at the 5′end of the transcript, suggesting a possibility of a wide repertoire of RNA caps in bacteria. In addition to NAD+ that had been shown to cap some of bacterial RNAs The crystal structure of promoter complex with NADpC dinucleotide transcript showed the interactions of nicotinamide moiety with the Rif-pocket of β subunit . Indeed,Km for NAD+ for promoters with −1C or −1T were lower compared to the promoters with −1G or A, the same tendency was observed for ATP, suggesting that the preference of NAD+ for −1 position is not explained by specific base-pairing of nicotinamide moiety with the −1 position of the template. We therefore suggest that −1 position of the promoter affects the properties of other steps(s) of initiation, thus affecting the efficiency of RNA NADylation.The crystal structure of the promoter complex with the NADpC revealed that nicotinamide moiety does not interact with the template . The aut70 versus stationary phase σS initiation factor. These results suggest that σ subunits do not directly interact with the initiating co-enzymes. We however cannot exclude that other alternative initiation factors can contribute towards or against incorporation of non-canonical capping nucleotides analogues. Furthermore, σ region 3.2 may affect the relative efficiency of capping by its interactions with the template DNA upstream of the start site, thus affecting escape into productive elongation.Region 3.2 of the σ subunit is in close proximity to the Rif-pocket  and may We further extended the potential bacterial RNA cap repertoire to derivatives of UDP, the highly abundant precursors of cell wall components. Interestingly we found that while σ region 3.2 did not have effect on the incorporation of UDG-Glc and UDP-GlcNAc, it did inhibit incorporation of a larger UDP-MurNAc pentapeptide. The protection of more complex cell wall precursor from incorporation into RNA may have biological significance. The more complex precursors are more expensive for the cell than initial precursors UDG-Glc and UDP-GlcNAc. Furthermore, modification of the 5′ end of RNA with UDP-MurNAc pentapeptide may potentially lead to targeting of an RNA to a membrane, thus affecting its expression.Km values for NAD+ are in the range of 100–400 μM on various promoter variants, which is below published intracellular concentration of 2.3 mM in exponential E. coli cells grown with glucose  on acnA promoter are also far lower the cellular concentration of these compounds   , and thus of UDP .There might be an unexpected connection between capping  and the effect of various antibiotics. For example, inhibition of protein synthesis by chloramphenicol or tetracycline, as well as inhibition of cell wall biosynthesis by fosfomycin lead to significant increase of the UDP-GlcNAc concentration , potentiin vitro.Overall our work provides mechanistic insight into the process of prokaryotic capping of RNA by RNAP with the variety of cellular cofactors


C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5:UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5:UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases  and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in  C. elegans. Here we report a new mechanism downstream of UNC-6/Netrin involving flavin monooxygenase redox enzymes (FMOs). We show that FMOs are normally required for axon guidance and to inhibit growth cone protrusion. Furthermore, we show that they are required for the anti-protrusive effects of activated UNC-40 and UNC-5 receptors, and that they can partially compensate for loss of molecules in the pathway, indicating that they act downstream of UNC-6/Netrin signaling. Based on the function of the FMO-containing MICAL molecules in Drosophila and vertebrates, we speculate that the FMOs might directly oxidize actin, leading to filament disassembly and collapse, and/or lead to the phosphorylation of UNC-33/CRMP, which we show also genetically interacts with the FMOs downstream of UNC-6/Netrin. In conclusion, this is the first evidence that FMOs might act downstream of UNC-6/Netrin signaling in growth cone protrusion and axon repulsion.Mechanisms that guide axons to their targets in the developing nervous system have been elucidated, but how these pathways affect behavior of the growth cone of the axon during outgrowth remains poorly understood. We previously showed that the guidance cue UNC-6/Netrin and its receptors UNC-40/DCC and UNC-5 inhibit lamellipodial and filopodial growth cone protrusion to mediate repulsion from UNC-6/Netrin in  The formation of neural circuits during development depends on the guidance of growing axons to their proper synaptic targets. This process relies on the growth cone, a dynamic actin based structure present at the tip of a growing axon. Growth cones contain a dynamic lamellipodial body ringed by filopodial protrusions, both important in guiding the axon to its target destination –4. GuidaThe secreted laminin-like guidance molecule UNC-6/Netrin mediates both axon attraction and axon repulsion and defines a dorsal-ventral guidance mechanism conserved from invertebrates to vertebrates –9. AttraC. elegans, UNC-6/Netrin is secreted by the ventral cells and along with its receptors UNC-40 and UNC-5 is required for the dorsal ventral guidance of circumferential neurons and axons ; LGIII: fmo-3(gk184651); LGIV: fmo-1(ok405), fmo-2(ok2147), lqIs128[Punc-25::myr::unc-40], unc-5(op468 and e152), unc-33(e204); LGV: fmo-4(ok294), fmo-5(tm2438), ehbp-1(ok2140M+); LGX: lqIs182[Punc-25::mig-2(G16V)]. Chromosomal locations not determined: lqIs129[Punc-25::myr::unc-40], lqIs296[Punc-25::myr::unc-5], lqIs204[Punc-25::ced-10(G12V)], lhIs6[Punc-25::mCherry], lqIs311[fmo-5 genomic] by integration of lqEx1047. Extrachromosomal arrays were generated using standard gonadal injection ; lqEx1014, lqEx1015, lqEx1016, lqEx1045, lqEx1046 and lqEx1047; lqEx949, lqEx950, lqEx951, lqEx1053, lqEx1054 and lqEx1055; lqEx1098, lqEx1099, lqEx1100, lqEx1101 and lqEx1102; lqEx952, lqEx953, lqEx954, lqEx1061, lqEx1062, lqEx1063, lqEx1078, lqEx1079 and lqEx1080; lqEx1146, lqEx1147, lqEx1148 and lqEx1149; lqEx1113 and lqEx1114; whEx28. Multiple (≥3) extrachromosomal transgenic lines of Pfmo-5::fmo-5 for overexpression data of fmo-5 were analyzed with similar effect, and one was chosen for integration and further analysis. Genotypes containing M+ indicate that homozygous animals from a heterozygous mother were scored. The ehbp-1(ok2140M+) strain was balanced with the nT1 balancer, since ehbp-1(ok2140) homozygous animals are sterile. RNAi was performed by feeding as previously described . Th. ThC. el protein . We anald ehbp-1  for VD/Dfmo-1, 4, and 5 and ehbp-1 mutants showed significant defects in VD/DD axon pathfinding, including ectopic axon branching and wandering  resembled ehbp-1 M+ mutants  strong loss-of-function mutants, most axons (92%) extended past the lateral midline despite wandering   . ehbp-1 e unc-40 .unc-5(e53) strong loss-of-function mutants display a nearly complete failure of VD axons to reach the dorsal nerve cord [unc-5(e152) is a hypomorphic allele [unc-5(op468) allele [fmo-1, fmo-4 and fmo-5 significantly enhanced the VD/DD axon guidance defects of both unc-5(e152) and unc-5(op468), but ehbp-1 did not  allele  also dis did not . While ffmo-1, fmo-4, and fmo-5 coding regions were driven in VD/DD motor neurons using the unc-25 promoter. Punc-25::fmo transgenes significantly rescued lateral midline crossing defects in fmo; unc-5(op468) and fmo; unc-5(e152)  . These dfmo-1 and fmo-5 promoter regions were active in intestinal cells and the excretory gland cell, whereas the fmo-4 promoter was active in hypodermal cells, duct and pore cells [ehbp-1 is expressed in all somatic cells including neurons [fmo-1, fmo-4 and fmo-5 were expressed in embryonic and adult neurons, including motor neurons [fmo-1, fmo-4, and fmo-5 to gfp. We could observe no fmo-1::gfp expression in transgenic animals, in contrast to previous studies using a LacZ reporter [fmo-1 . Our fmo-1::gfp transgene might be missing regulatory regions required for expression. fmo-4::gfp was expressed strongly in hypodermal cells, excluding the seam cells and vulval cells, consistent with previous studies [fmo-4::gfp expression in cells in the ventral nerve cord fmo-1 andrve cord . Pfmo-5:reported  Fig 6E)fmo-1 andfmo-1, fmo-4, and fmo-5 mutant growth cones displayed longer filopodial protrusions compared to wild type (e.g. 0.96 µm in wild type compared with 1.55 µm in fmo-5(tm2438); p < 0.001) (ehbp-1(ok2140)  also display ectopic branches, likely due to failure of filopodial retraction and subsequent consolidation into a neurite [The growth cones of dorsally-directed VD commissural axons are apparent in early L2 larvae . We imag< 0.001) . This ef(ok2140) . Growth  mutants , as othe neurite , 17.fmo-5 coding region driven in VD/DD motor neurons using the unc-25 promoter also significantly rescued axon guidance defects as well as the long filopodial protrusions seen in fmo-5(tm2438)  . We exprpromoter  and obsemyr::unc-40 and myr::unc-5) results in small growth cones with few if any filopodial protrusions (i.e. protrusion is constitutively inhibited by MYR::UNC-40 and MYR::UNC-5) [fmo-1, fmo-4, fmo-5 and ehbp-1 significantly suppressed inhibition of filopodial protrusion and growth cone size caused by myr::unc-40  , 17, 18.::unc-40  and myr:r::unc-5 . Notablyr unc-40 , suggestfmo-1, fmo-4 and fmo-5 suppressed filopodial protrusion deficits caused by ced-10(G12V) and mig-2(G16V) (ehbp-1 suppressed mig-2(G16V), but ehbp-1(ok2140M+); ced-10(G12V) double mutants were inviable and could not be scored. Furthermore, fmo-4 and fmo-5, but not fmo-1, significantly suppressed growth cone size reduction caused by CED-10(G12V) and MIG-2(G16V). ehbp-1 also suppressed growth cone size reduction of MIG-2(G16V). Taken together, these data indicate that functional FMO-1, FMO-4, FMO-5, and EHBP-1 are required for the full effect of MYR::UNC-40, MYR::UNC-5, CED-10(G12V), and MIG-2(G16V) on growth cone protrusion inhibition, including filopodial protrusion and growth cone size.Expression of activated CED-10(G12V) and MIG-2(G16V) in the VD neurons results in reduced growth cone protrusion similar to MYR::UNC-40 and MYR::UNC-5 . We foun-2(G16V) . ehbp-1 fmo-5 loss-of-function mutant growth cones displayed excessively-protrusive filopodia (fmo-5(tm2438) mutant VD growth cones  background, possibly due to the decreased levels of FMO-5 compared to the wild-type background.ilopodia  and suppilopodia –11. Tranth cones . In a wiilopodia , indicatunc-5, unc-73, and unc-33 result in excessively large growth cones with increased filopodial length , unc-73(rh40), and unc-33(e204)  (fmo-5 transgenic expression with unc-33(e204) could also indicate that FMO-5 and UNC-33 represent distinct, compensatory pathways downstream of UNC-5 and the Rac GTPases to inhibit filopodial protrusion. unc-33; fmo-5 double mutants did not show any significant increase in filopodial length  . Howevern alone) . These dl length , which wunc-5 mutants, unc-40 single mutants display shortened filopodial protrusions and a relatively normal growth cone size  suppressed growth cone area and filopodial length of fmo-1(ok405) mutants  domain, a LIM domain and multiple CC domains. No molecule encoded in the C. elegans genome has a similar multi-domain organization. However, the Eps-15 homology (EH) domain binding protein EHBP-1 is similar to the non-FMO portion of MICAL and contains a CH domain [C. elegans does not have a multidomain MICAL-like molecule, it is possible that the functional equivalents are the FMOs and EHBP-1.Results here implicate the guidance . The MICguidance , 45. HerH domain . We showfmo-1, fmo-4, fmo-5, and ehbp-1 mutants display defects in dorsal guidance of the VD/DD motor axons that are repelled from UNC-6/Netrin  rescued VD/DD axon guidance defects , whereas expression from the hypodermal dpy-7 promoter did not . We speculate that the FMOs are but one of several pathways mediating the effects of UNC-40 and UNC-5 in axon pathfinding. ehbp-1 did not enhance unc-40 or unc-5, suggesting discrete roles of these molecules or wild-type maternal ehbp-1 contribution. fmo-1, fmo-4, fmo-5, and ehbp-1 mutations each suppressed the effects of activated MYR::UNC-40 and MYR::UNC-5 on inhibition of growth cone protrusion (fmo-5 transgenic expression suppressed unc-5(e152) growth cone area and filopodial protrusions (Our findings suggest that the FMOs act with the UNC-40 and UNC-5 receptors to mediate UNC-6/netrin repulsive axon guidance and inhibition of growth cone protrusion. otrusion . In thistrusions . That thunc-5 are much stronger than those of the fmo mutants). One explanation for this is that these molecules act in networks rather than simple linear pathways. The FMOs might be one of many mechanisms acting downstream of UNC-5, and multiple pathways might converge on UNC-33 (e.g. UNC-5 and UNC-33 are major “nodes” in this network). Further loss- and gain-of-function studies will be required to understand this signaling network.Interestingly, mutations in these genes have very distinct penetrances  and MIG-2(G16V)    and MIG-2(G16V) inhibit VD growth cone protrusion. We show that mutants) . Furthern alone) . UNC-73/otrusion . That FMC. elegans CRMP-like molecule UNC-33 is required in a pathway downstream of Rac GTPases for inhibition of growth cone protrusion in response to UNC-6/Netrin [unc-33 loss-of-function mutants with FMO-5 transgenic expression displayed a mutually-suppressed phenotype. The excessively-long filopodial protrusions of unc-33 mutants were reduced to wild-type levels, but were significantly longer than in animals with FMO-5 transgenic expression, and the growth cone area was reduced to resemble FMO-5 transgenic expression alone  Hof NADPH . Upon accollapse . Thus, tC. elegans flavin-containing monooxygenase molecules (FMOs) in inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling. The FMOs acted downstream of the UNC-6/Netrin receptors UNC-5 and UNC-40, and downstream of the Rac GTPases CED-10 and MIG-2 (In summary, we present evidence of a novel role of the nd MIG-2 . Future


A 2 °C increase in global temperature above pre-industrial levels is considered a reasonable target for avoiding the most devastating impacts of anthropogenic climate change. In June 2015, sea surface temperature (SST) of the South China Sea (SCS) increased by 2 °C in response to the developing Pacific El Niño. On its own, this moderate, short-lived warming was unlikely to cause widespread damage to coral reefs in the region, and the coral reef “Bleaching Alert” alarm was not raised. However, on Dongsha Atoll, in the northern SCS, unusually weak winds created low-flow conditions that amplified the 2 °C basin-scale anomaly. Water temperatures on the reef flat, normally indistinguishable from open-ocean SST, exceeded 6 °C above normal summertime levels. Mass coral bleaching quickly ensued, killing 40% of the resident coral community in an event unprecedented in at least the past 40 years. Our findings highlight the risks of 2 °C ocean warming to coral reef ecosystems when global and local processes align to drive intense heating, with devastating consequences. We developed a reef-flat heat budget to diagnose the physical drivers of warming in June 2015, linking it to a regional atmospheric anomaly, and tracked the coral community response to extreme thermal stress. Finally, we evaluate the historical precedence for an event of this magnitude using markers of thermal stress preserved in the skeletons of long-lived corals.The ocean surface surrounding coral reefs in the northern SCS has warmed at a rate of 0.09 ± 0.015 °C/decade (95% confidence) since 1900 , closely202526in situ logger on the reef flat during June-July-August (JJA) of 2013–2015 was 29.7 °C, practically identical to that of the surrounding open ocean  (Dongsha Atoll is a massive (25 km diameter), circular coral reef emerging from ~500 m water depth on the continental shelf slope in the northern SCS. The living reef flat encircling the lagoon is just 1–3 m deep , as is cf Watch) . Howeverf Watch) . As a ref Watch) , disruptf Watch) . For sevf Watch)  and 3. Wf Watch) . The extEcological surveys conducted across the reef flat in early June prior to the bleaching event, and again in late July provide a rare quantitative characterization of the response of the benthic community to the extreme thermal stress28In contrast to the reef flat, no bleaching was observed on the upper fore reef slope or in the channel north of Dongsha Island, where local amplification of warming did not occur. Large-amplitude internal waves are generated on tidal frequencies in the Luzon Strait to the east of Dongsha Atoll and propagate along the thermocline (70–100 m depth) into the northern SCS1717Porites, Acropora, Pavona, and Stylophora, the four most common coral genera on the reef flat, appeared bleached. However, six weeks later, we found only 17% mortality of Porites in our ecological point-count data, compared with 56% for Acropora  of these colonies died by late July. Average growth rate was 1.5 cm yr−1  resolution, NOAA-OI begins in 1982 at 1° resolution, Coral Reef Watch begins only in 2013 at high resolution (5 km), and MODIS covers only 2002-present but at very high spatial resolution (4 km). ERSST and NOAA-OI anomalies were calculated relative to the 1940–1970 ERSST mean. The NOAA Coral Reef Watch program calculated monthly climatologies for 1985–2012, and we used this as the climatology in our assessment of SST anomalies in the open ocean around Dongsha Atoll during June 2015. ERSST data were used to evaluate the centennial-scale warming trend in the northern South China Sea (i.e. MODIS data were used only to make Sea surface temperature (SST) data were acquired from the Extended Reconstructed SST (ERSST) producthina Sea . MODIS wThe physical conditions leading to the bleaching event were monitored with a series of instruments deployed on Dongsha Island and underwater on the reef flat . Solar rT) change by: t is time, Q is heat flux in W m−2, ρ is seawater density (kg m−3), cp is the heat capacity of seawater (W s kg−1 °C−1), and h is water depth (m). The total heat budget (in °C hr−1) on the reef flat was determined by taking the time-derivative of measured water temperature, and the advective component was estimated as the difference between total (observed) and air-sea  components (benthic and diffusive heat fluxes assumed negligible).We used the Dongsha Island meteorological data to estimate the air-sea heat flux in June 2015 as the sum of latent, sensible, longwave radiation, and shortwave radiation fluxes . LongwavEcological surveys were conducted at seven stations across the reef flat and two stations on the fore reef following a protocol similar to previously established methods for characterizing benthic cover on coral reefsPorites colonies using underwater pneumatic drills with 3 cm diameter drill bits. The cores were scanned at Woods Hole Oceanographic Institution Computerized Scanning and Imaging Facility and skeletal density was calculated by comparison to previously calibrated coral skeletal density standards404142Coral skeletal cores were collected from massive How to cite this article: DeCarlo, T. M. et al. Mass coral mortality under local amplification of 2 °C ocean warming. Sci. Rep.7, 44586; doi: 10.1038/srep44586 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Leishmania spp. and Trypanosoma brucei and found evidences for its expression as a specific RNA class. The trypanosomatid TERRA are heterogeneous in size and partially polyadenylated. The levels of TERRA transcripts appear to be modulated through the life cycle in both trypanosomatids investigated, suggesting that TERRA play a stage-specific role in the life cycle of these early-branching eukaryotes.The Telomeric Repeat-containing RNAs (TERRA) participate in the homeostasis of telomeres in higher eukaryotes. Here, we investigated the expression of TERRA in  Trypanosoma brucei, a process that allows evasion from the host immune system during infection (Li 2015). Despite the growing body of information on trypanosomatid telomere biology , our knowledge of TERRA transcription and function in these early-branching eukaryotes remains limited. Here, we report the detection of TERRA transcripts in Leishmania spp. and T. brucei; analyse the structure of the 5’ and 3’ ends of TERRA molecules; and present evidence for modulated TERRA expression through the life cycle of these trypanosomatids.Telomeres of yeast and mammalian chromosomes are transcribed into long non-coding RNAs, known as Telomeric Repeat-containing RNAs (TERRA) . In mammals, RNA polymerase II-driven transcription of TERRA proceeds from within subtelomeric regions towards the chromosome ends and at least a proportion of TERRA molecules contains a 5’-end 7-methylguanosine cap and a 3’-end poly(A) tail . Recent advances in the understanding of TERRA function has indicated its participation in telomere homeostasis by modulating telomerase activity, recruiting chromatin modifying factors and also by mediating the capping of chromosome ends . Similar to other eukaryotes, the linear chromosomes of trypanosomatids are capped by telomeres composed of TTAGGG repeats and participate not only in protection of chromosome ends, but also in the control of expression of subtelomeric genes . Subtelomeric and telomeric regions are pivotal for antigenic variation in Detection of TERRA in Leishmania - To investigate the potential transcription of telomere repeats in different Leishmania species, Northern blots of total RNA were initially probed with the oligonucleotides (CCCTAA)15 and (TTAGGG)15 that allow the distinction between sense (UUAGGG)n and antisense (CCCUAA)n transcripts, respectively , who found that the transcription of telomeres in different kinetoplastid species was predominantly unidirectional and proceeded from a chromosomal internal region towards the chromosomal end.ectively . The senLeishmania major .To investigate whether the unidirectional TERRA transcript resulted from readthrough transcription of subtelomeric-located genes, we used reverse transcription-polymerase chain reaction (RT-PCR) to analyse the transcription at specific telomere ends of ia major . The revia major , which wL. braziliensis are polyadenylated. By using RT-PCR analysis we were able to detect TERRA transcripts in cDNA that was synthetised using oligo(dT) as primer . It is noteworthy that TERRA species of ~1.6, ~2.1 and ~3.1 Kb were enriched in the poly(A)+ fraction  treatment, which preferentially degrades 5’-mono-phosphorylated RNA. Northern blot analysis of total RNA did not reveal significant decrease in TERRA signal upon TE treatment  protocol to purify metacyclic parasites from stationary-phase promastigotes of L. major . We then performed Northern blot analysis with RNA prepared from procyclic stationary phase parasites (PNA+) and metacyclic parasites (PNA-) and observed that the levels of TERRA transcripts were elevated in the RNA samples from PNA- parasites  and procyclic (PC) forms (Leishmania and as previously reported (Rudenko & Van der Ploeg 1989), the TERRA transcripts were detected as a smear between 0.5 and 10.0 Kb in both BS and PC forms. Notably, TERRA levels were substantially higher in BS forms when compared to PC forms, suggesting that T. brucei TERRA is differentially expressed between these two life cycle stages.C) forms . SimilarL. braziliensis, RT-PCR analysis indicated that TERRA is also polyadenylated in T. brucei BS forms , which could be due to differences in parasite cell lines. We also analysed the susceptibility of TERRA from T. brucei BS forms to TE treatment. As shown in T. brucei possess a di or tri-phosphorylated or a CAP structure at the 5’ end.Similar to BS forms . Also, f. brucei . This isT. brucei RNA samples for antisense transcripts (CCCUAA)n we detected a distinct transcript of ~1,0 Kb in PC forms that was absent in BS forms n-containing transcript  cell line. As shown in Surprisingly, when we probed the BS forms . This transcript . In addiL. major and T. brucei. Further studies are required to dissect the underlying biological mechanisms leading to the differential processing of 5’ end of TERRA of L. braziliensis relative to T. brucei. Perhaps this is the result of the different RNA polymerases involved in TERRA biogenesis in the two parasites. Also, the role of TERRA in the maintenance and functioning of the trypanosomatid genome and of the antisense telomere-sequence containing RNA expressed in procyclic forms of T. brucei should also be the subject of further investigation.Our findings suggest that, similar to other eukaryotes, TERRA is a distinct transcript class in trypanosomatids. The initial characterisation we present here suggested that TERRA levels seem to be controlled throughout the life cycle of