High-altitude environments dramatically influenced the genetic evolution of vertebrates. However, little is known about the role of RNA editing on high-altitude adaptation in non-model species. Here, we profiled the RNA editing sites (RESs) of heart, lung, kidney, and longissimus dorsi muscle from Tibetan cashmere goats  and Inner Mongolia cashmere goats  to reveal RNA editing-related functions of high-altitude adaptation in goats. We identified 84,132 high-quality RESs that were unevenly distributed across the autosomes in TBG and IMG, and more than half of the 10,842 non-redundant editing sites were clustered. The majority (62.61%) were adenosine-to-inosine (A-to-I) sites, followed by cytidine-to-uridine (C-to-U) sites (19.26%), and 32.5% of them had a significant correlation with the expression of catalytic genes. Moreover, A-to-I and C-to-U RNA editing sites had different flanking sequences, amino acid mutations, and alternative splicing activity. TBG had higher editing levels of A-to-I and C-to-U than IMG in the kidney, whereas a lower level was found in the longissimus dorsi muscle. Furthermore, we identified 29 IMG and 41 TBG population-specific editing sites (pSESs) and 53 population-differential editing sites (pDESs) that were functionally involved in altering RNA splicing or recoding protein products. It is worth noting that 73.3% population-differential, 73.2% TBG-specific, and 80% IMG-specific A-to-I sites were nonsynonymous sites. Moreover, the pSESs and pDESs editing-related genes play critical functions in energy metabolisms such as ATP binding molecular function, translation, and adaptive immune response, which may be linked to goat high-altitude adaptation. Our results provide valuable information for understanding the adaptive evolution of goats and studying plateau-related diseases. GluR2 transcript revealed that the substrate of ADAR2 is crucial for the normal development of the nervous system of zebrafish, ADAR1-deficient mice died around 12 days after birth, and ADAR1 deficiency caused A-to-I editing depletion at the A and B sites of 5-HT2C receptor transcripts. [The functions and mechanisms of RNA editing have been well studied, the functions of which include modifying the sequence of RNA precursors ,2, changscripts. .SMAD3, SIAH1, and CDK13 had embedded functional editing sites and were involved in muscle development [RNA editing is affected by many factors, including temperature, tissue context, genotype, feeding conditions, and age ,15, whilelopment . Furtherelopment ,20,21,22elopment , sheep [elopment , and goaelopment . For inselopment , which mThe species’ adaptation to local conditions has been promoted through generational inheritance . In partWe identified an average of 5747 candidate RESs for 40 samples. After filtering the raw RNA editing sites, 84,132 high-quality RESs were identified , and 10,More than half (5920) of the non-redundant sites were detected in at least five samples, with 35 sites (0.32%) appearing in 40 samples A. The edADAR, ARARB1, ADARB2, APOBEC1, APOBEC2, and APOBEC4 in goats. A total of 724 (32.5%) RESs significantly correlated with the expression of catalyzing genes, and 90.5% of them positively correlated, whereas 38.4% of C-to-U sites negatively correlated with the expression of APOBEC2  were strongly correlated  and the expression levels of catalyzing genes such as  APOBEC2 A. Among ectively . This firrelated . The corrrelated B.To determine whether RNA editing sites are enriched in specific sequence motifs, we explored the base preference of RESs (1389 and 838 non-redundant A-to-I and C-to-U editing sites) and found a differing probability for their 10-bp flanking region  C. For thTo investigate RNA editing involved in alternative splicing of gene transcripts, 1228 RESs were evaluated in 867 exons of 849 transcripts from 552 genes . The disTo systematically investigate the RNA editing profiling of four tissues  from TBG and IMG, we generated a pairwise comparison across tissues and populations based on 1389 A-to-I and 838 C-to-U non-redundant editing sites . The A-tRESs specifically appearing in organs involve a tissue-specific process. We investigated the function of the tissue-specific RNA editing sites (tSESs). As shown in SLC5A12, SLC13A1, and SLC22A4) were significantly enriched in symporter activity and ion transport, which play a crucial role in maintaining homeostasis. In comparison, five muscle-related genes  function in contractile fiber and sarcomere   and population-differential editing sites (pDESs) to define the function of RESs in population adaptation. The majority (78.1%) of A-to-I and C-to-U sites were shared between TBG and IMG, whereas 10.6% and 11.4% in IMG and TBG, respectively A. Then, 7, etc.) . Of them7, etc.) C, and 697, etc.) D. Additi7, etc.) E. The ed7, etc.) F–H. Only process I. We fur process . These rRNA editing plays a critical role in all aspects of RNA transcripts, including splicing, mRNA stability, and translation. A-to-I RNA editing, which is the most prominent RNA editing type ,22, has APOBEC1 [We systematically identified the RNA editing events in goats and obtained 84,132 high-quality RESs, their non-redundant sites 10,842) were distributed unevenly among the chromosomes, which is consistent with the study in bovine and yak ,42. In a were disAPOBEC1 , which cAPOBEC1 . This inADAR1 and APOBEC1 play vital roles in RNA editing and transcript stability [AIMP2 [Pin1, and WWP2 [ADAR family, and C-to-U editing is primarily catalyzed by APOBEC1 [APOBEC2 until now. We could not draw a reliable conclusion about the correlation between the editing levels of RESs and the expression levels of RNA editing catalyzing genes among tissues because no significant trends were observed. This may be caused by the following factors: (1) We found no obvious tissue-specific manner of RNA editing catalyzing genes (except APOBEC4) in the heart, kidney, lung and longissimus dorsi; (2) RNA editing is affected by other regulators, but we found less tSESs; (3) the limited of tissue types. It is worth noting that in our research, C-to-U editing sites correlated with the expression of APOBEC1 and APOBEC2, which amounted to 94 and 104, respectively, suggesting that APOBEC2 may have editing activities. Since RNA editing may be relevant to editing-related genes [SFTPB [ATP2A1 [EIF2AK4 [KLF13 [LDB3 [SLC13A1 [ACMSD [RNA editing events were mediated by their editing catalyzing genes and related to editing-related gene expression. Previous studies show that tability , and thetability ,46,47. Iy [AIMP2 , Pin1, aand WWP2  or otherand WWP2 . In addi APOBEC1 ,52 becaued genes , we founs [SFTPB ,54, ATP2 [ATP2A1 , EIF2AK4[EIF2AK4 , KLF13 [4 [KLF13 , LDB3 [513 [LDB3 , SLC13A1[SLC13A1 , and ACM1 [ACMSD , with orAdditionally, the specific sequence around RNA editing sites might have an impact on the RNA structure and its editing patterns. In accordance with previous studies carried out in chickens , pigs 2, bovinesComparing RNA editing profiles across tissues  from TBG and IMG, we realized there were little differences in the distribution of A-to-I and C-to-U editing sites between TBG and IMG, which come from a common ancestor. Consistent with a previous study, more A-to-I and C-to-U sites were edited in the lung 1669) and kidney (1526) tissues and fewer in the muscles (heart 1221 and longissimus dorsi 1419) . In addi9 and kidMYH8, MYH2, MYH3, NEB, and OBSCN) were significantly enriched in muscle functions. These results confirm that tissue-specific RNA editing patterns and functions exist in goats.RNA editing sites vary across tissues, such as A-to-I events, which are dominantly tissue-dependent ,65. Our ZNF644 [ATF6 [ADAM10 [MYCT1 [OBSCN [OGFOD1 [MTO1 [ARAP2 [SCP2 [KIF17 potentially regulate the body’s homeostasis to adapt to environmental stress [By characterizing the functional aspect of RESs, we anchored 41 and 29 population-specific editing sites (pSESs) in TBG and IMG, respectively, and 53 population-differential editing sites (pDESs). Their corresponding genes, such as ZNF644 , ATF6 [744 [ATF6 ,72, and  [ADAM10  in TBG, 0 [MYCT1 , OBSCN [1 [OBSCN , and OGF [OGFOD1  in IMG, D1 [MTO1 ,79, ARAP1 [ARAP2 , SCP2 [8P2 [SCP2 , and KIFl stress . We alsol stress ,84,85. F2 content), we analyzed the origin, performances , and genetic distances among goat populations. Finally, the IMG living in Alxa of Inner Mongolia grassland  were anchored since they performed closely and shared a common ancestor with TBG [To attain an excellent experimental control for the high-altitude TBG living in Rutog county of the Qinghai-Tibet Plateau . Then, a high-throughput sequencing library with an average length of 80 insert fragments of 350 bp was constructed for DNA sequencing by double-terminal 10× with a 150 bp reading length using an Illumina HiSeq platform sequencing instrument .Total RNA was extracted from tissue samples using the Trizol method  and then quantified by a NanoPhotometer spectrophotometer . The mRNAs enriched by polyA magnetic beads from qualified RNA samples were used to construct the library. Finally, an Illumina HiSeq 2500 sequencing platform  was employed for double-terminal 300 bp (2 × 150 bp) high-pass sequencing, and clean reads amounted to 10 G per library.Trimming: Genomic DNA and RNA raw data were analyzed using Trimmomatic v0.39 to filter connectors and remove readings containing more than 10% unknown bases or reading segments containing more than 50% low-quality (Qphred ≤ 20) bases.Alignment: Using BWA v0.7.17, we aligned DNA clean reads to the ARS1 goat reference genome (refgenome) retrieved from an Ensembl database (release-95). In addition, RNA clean reads were mapped to the same refgenome by HISAT2 v2.1.0 after generating an index on the goat refgenome ARS1 using hisat2-build. It was based on the known gene annotation file , extracting the splicing sites and exons coordinated from refgenome by Python scripts extract_splice_sites.py and extract_exons.py with default parameters. Then we used default-set hisat2 to align the sequence and refgenome.BAM process: DNA and RNA alignment BAMs were sorted by SAMtools v1.8.0 with their genome coordinates. To avoid potential PCR or sequence optical artifacts, we removed duplicated reads mapped to the exact location by the MarkDuplicates tool in GATK v4.1.8.0. FixMateInformation function in GATK was further used to ensure that all matching sequencing read paragraphs were synchronized.Variation calling: To reduce the random systematic error caused by sequencing instruments, we retrieved goat single nucleotide polymorphisms (SNPs) from published databases, including Ensemble (release-95) and variant call format (VCF). We then combined them using BaseCalbrator and ApplyBQSR in GATK v4.1.8.0 for each sequence alignment file by recalibrating the base quality score. Then, HaplotypeCaller in GATK was used to call variation, and SelectVariants was employed for separating SNP and short fragment insertion and deletion (INDEL). No filter process was performed in this step to obtain the complete genomic variation results, allowing the strictest control of subsequent RESs identified.RESs Detection: Using REDItools v1.2.1 , we deteRESs Filtration: The selectPositions.py in REDItools was firstly used to filter candidate RESs with default parameters, including -c 10 -C 5 -v 2 -V 0 -f 0.01 -F 0.95 -e -u, denoting that: (1) RNA sequence reads covered that site at least ten times; (2) DNA sequence reads covered that site at least five times; (3) Minimum support reads of editing base is 2; (4) The number of bases supporting DNA sequence mutations is 0; (5) Minimum mutation proportion of RNA sequence is 0.01; (6) Minimum non-mutant proportion of DNA sequence is 0.95; (7) Eliminates multiple base mutations at the same site. Moreover, the unqualified candidate RESs were excluded with the following conditions: (1) Overlapped with the known SNPs or INDELs retrieved from the Ensemble database (release-95) via intersectBed function of BEDtools v2.17.0; (2) Detected in less than three samples; (3) With editing level amounted to 1 in all detected samples; (4) Located at sex chromosomes or unplaced in refgenome. All downstream analyses were based on the obtained high-quality RESs.Note: The RNA sequencing library in this study was a ploy(A) strategy and non-strand specific. Thus, we cannot precisely determine which strand occurred in RNA editing events. Therefore, both AG bases change that appeared in positive-stranded genes and TC in reverse-stranded genes reported by REDItools were regarded as A-to-I editing. Similarly, CT in positive-stranded genes and GA in reverse-stranded ones were assigned as C-to-U editing.www.uniprot.org, accessed on 12 March 2021), a high-quality database of protein sequences and functional information (UniProt 2021). Ensembl gene IDs were converted to Entry IDs to obtain their homologous proteins’ functions. STRING  was used for constructing protein-protein interaction networks based on human annotation.Based on the goat ARS1 genome annotation file , the intersectBed function of BEDtools v2.27.1 was employed to intersect RESs to gene feature regions, including exons, introns, 5′-UTR, 3′-UTR, and intergenic regions. To investigate the functions of candidate RESs genes, we performed Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using the Fisher test, based not on the poorly annotated goat genome but on the human database (org.Hs.eg.db v 3.14.0). Meanwhile, the functions of those Ensembl genes without official symbols were investigated through UniProt  algorithm, and the significance level was set at 0.05.All R package ggseqlogo was used to plot the frequency of bases. A total of 21-bp sequences containing each RES and its flanking 10-bp upstream and downstream region in the goat genome were extracted. We selected editing sites in CDSs to obtain the codon sequence and counted the distance from the start codon after removing the non-coding region. We then divided the accepted coding length by 3, to attain the codon’s position. Codons coded the edited amino changed A to G or C to T at those positions. For those RESs located in the negative sense, we converted them to complementary antisense sequences.p < 0.05. With five samples collected per tissue, the RESs exclusively appeared in only one tissue and were defended as tSESs.Tissue-specific editing sites (tSESs): To begin, we compared RESs in four organs collected from each goat individual. Then, ANOVA in R v4.0.2 was used to compare the difference in editing level between tissues, and the significance threshold was set to p-values were adjusted via the padjust function in R v4.0.2 with Benjamini and Hochberg’s (BH) algorithm, and the threshold was set to 0.05.Population-differential editing sites (pDESs): We filtered out high-quality RESs supported by at least three samples in a tissue per population to compare the RNA editing profiling between populations unbiasedly. Then pDESs were defined as those RESs passing the Wilcox test, representing significantly different editing levels between groups in the same tissue. The Population-specific editing sites (pSESs): Those sites altered in a specific group with at least three samples supported per tissue were defined as pSESs. For example, if an RNA editing event appeared in the kidney of three IMG individuals but failed to be identified in the TBG samples, we defined it as an IMG pSESs in the kidney.p-values < 0.05 and log2 fold change (log2FC) > 1 as a significant threshold.We quantified the gene expression using stringtie v1.3.4d for calculating read counts based on alignment BAM files and a genome coordinate file for each gene characteristic . After generating the gene count matrix, we normalized data using CalcNormFactors and cpm functions in the R v4.0.2 package edgeR v3.38.2 and then calculated gene expression using RPKM (Reads Per Kilobase Million) method. Differential expression genes were detected using the exactTest process in edgeR v3.38.2, with BH adjusted p-value < 0.05.The correlation between RNA editing level and the expression of its editing-related gene for each RESs was detected for 40 individuals using the R function correlation (cor) with the Spearman method and significance threshold set to We identified thousands of RESs in the heart, lung, kidney, and longissimus dorsi muscle from TBG and IMG and characterized their potential functions, such as recoding protein and alternative splicing. Furthermore, our findings revealed that the majority tSESs, pSESs, and pDESs showed adaptive signals in the tissues and populations. This will be helpful to animal breeders and biomedical researchers studying plateau-related diseases.


Although 25% of the Ethiopian population is young, Sexual and Reproductive Health (SRH) Services have not been adequately researched and intervened, leaving adolescents with many reproductive health challenges. Assessment of the uptake of each element of SRH service and its determinants among those age groups is essential to improving service uptake and reducing the burden of illness and disability in adolescents. Thus, this study aimed at assessing the uptake of key elements of SRH services and its determinants among adolescents residing in rural districts of Guraghe zone, Southern Ethiopia.A community-based cross-sectional study was carried out from May 1 to 30, 2020, and a multi-stage sampling technique was employed to randomly select 1028 adolescents. The data were collected by using a pre-tested interviewer-administered questionnaire. The data were coded and entered into Epi-Data version 3.1 and exported into SPSS version 23 for analysis. Independent t-tests and analysis of variance (ANOVA) were run to determine whether there were statistically significant differences in the mean number of SRH services used across each categorical variable. A multivariable generalized linear regression (GLM) model with a Poisson link was used to determine the effect of each variable on the mean number of SRH services used. Adjusted odds ratios with their corresponding 95% confidence interval were used to declare the statistical significance of the independent variables.The study included 1,009 adolescents, yielding a response rate of 98.1%. The use of the SRH service was assessed using eight elements, and the mean (± SD) score of service uptake was 4.05 (± 1.94), with only 6.8% of adolescents receiving all key elements. Comprehensive sexuality education (55.1%) and voluntary HIV/AIDS counseling and testing (51.0%) were the commonest service items used by adolescents, while the provision of contraceptives was the lowest service item received (25.9%). Educational level , having a parental discussion , lack of youth clubs , and knowledge on SRH issues  were identified as significant predictors of the uptake of key elements of SRH services.The overall uptake of SRH services was found to be low in the study area. Schools should be an excellent means of educating adolescents to increase their knowledge of key elements of SRH services. Furthermore, stakeholders must work together to improve the culture of parental discussion with adolescents and establish and strengthen youth clubs, as measures for encouraging the use of SRH services. Progress towards universal health coverage requires keeping adolescents healthy, as adolescence represents a significant opportunity for successful prevention and health promotion with life-course implications. Sexual and reproductive health (SRH) service is well described as the combination of techniques, procedures, and services that prevent and respond to sexual health snags by ensuring sexual health and well-being. While most adolescent health problems are avertible or fixable, adolescents face many hurdles in accessing health care and information, especially those who are in rural corners. Although 25% of the Ethiopian population is young, Sexual and Reproductive Health (SRH) Services have not been adequately researched and intervened, leaving adolescents with many reproductive health challenges. Assessment of the uptake of each element of SRH service and its determinants among those age groups is essential to improving service uptake and reducing the burden of illness and disability in adolescents. Thus, this study aimed at assessing the uptake of key elements of SRH services and its determinants among adolescents residing in rural districts of Guraghe zone, Southern Ethiopia. A multi-stage method of sampling was employed, and adolescents have been randomly chosen and participated in the study. Pre-tested structured questionnaires were used for data collection. Statistical significance between dependent and independent variables was checked by using bivariate analysis across ANOVA and independent t-tests. To see the weight of each explanatory variable on SRH service utilization, a generalized linear regression model (GLM) with a Poisson link was done. The overall uptake of SRH services was found to be low in the study area. Comprehensive sexuality education (55.1%) and voluntary HIV/AIDS counseling and testing (51.0%) were the commonest service items used by adolescents, while the provision of contraceptives was the lowest service item received (25.9%). Educational level, having a parental discussion, lack of youth clubs, and knowledge of SRH issues were identified as significant predictors of the uptake of key elements of SRH services. Schools should be an excellent means of educating adolescents to increase their knowledge of key elements of SRH services. Furthermore, stakeholders must work together to improve the culture of parental discussion with adolescents and establish and strengthen youth clubs, as measures for encouraging the use of SRH services. Adolescents are people between the ages of 10 and 19, According to the World Health Organisation (WHO) . The SexThere are approximately 1.2 billion adolescents globally. Adolescents constitute up to a quarter of the population in some countries, and the number of adolescents is expected to increase by 2050, particularly in low- and middle-income countries (LMICs), where access to health and social services, jobs, and livelihoods appears to be under pressure , 9. In sDespite international initiatives emphasizing adolescents' rights to SHR services, neither the providers of these services nor the systems in which they operate are equipped to meet the needs of adolescents in developing countries –13. MostThe adoption of sexual and reproductive health (SRH) services among adolescents have not been adequately researched or implemented, despite the fact that 23% of the SSA population was adolescent . Up to 6According to the 2019 Mini Ethiopian Demographic and Health Survey (2019 mini EDHS), contraceptive use in currently married women aged 15–19 was only 36.5%, with 27.5%, 5.9%, and 0.0% injectables, implants, and IUDs respectively. At 75% Adolescents must be kept healthy to progress toward universal health coverage, as adolescence represents a critical opportunity for the successful future of the nations . For eveIt is critical to assess the level and determinants of the use of key SRH services in this age group in order to increase uptake and reduce the burden of preventable morbidities and mortalities. Furthermore, understanding and appreciating the pattern of SRH service utilization among adolescents will aid in future planning for better service delivery by increasing knowledge and skills and providing a safe and supportive environment . Most st. There were 128 health facilities in total, which included 74 health centers, 5 hospitals, 168 health posts, and 30 private clinics.A community-based cross-sectional study was conducted in the Gurage Zone, central Ethiopia from May 1 to 30, 2020. The area is located 158 km from Addis Ababa  and 337 km from Hawassa . The adolescent population (15–19 yrs) accounted for 13.6% of the total population. The area is divided into 11 administrative districts encompassing 174 rural kebeles All adolescents in rural districts of the Guraghe zone were the source population, and all adolescents in the selected districts in the zone during the study period were the study population. The study excluded participants who have lived in the study area for less than six months and adolescents who were seriously ill at the time of data collection.Using the single population proportion formula, the sample size was determined by considering the following parameters: the proportion of 41.2%  who lived outside of the study area, and changes were made based on the results of the pre-test to improve responses to the questionnaires. The reliability of the questionnaires was assessed by SPSS using the reliability index for practical questions , which was 0.77. continuous supervision was done throughout the data collection period. The data collected were checked for completeness and inconsistencies before analysis.The data were entered into EpiData version 3.1 and exported to SPSS version 23 for analysis. Using descriptive statistical analysis, the frequency, percent, and mean of explanatory and response variables were computed. By using bivariate analysis through ANOVA and independent t-tests, statistical significance was tested between dependent and independent variables. In bivariate statistical analysis (ANOVA and Independent t-test), variables with p-value ≤ 0.05 was considered as candidate for multivariable statistical analysis . The determinants of the use of SRH services have been established using the Generalized Linear Model (GLM) technique. The Poisson regression analysis was carried out because our outcome variable of interest was calculated in terms of counting variables. It satisfies the equidispersion assumption as it tests the Poisson regression model assumption. FinallySexual and reproductive health (SRH): is a constellation of services comprising: consultation and provision of modern contraceptives; counseling and provision of safe abortion care; prevention and care of STIs; prevention and care for HIV/AIDS; provision of comprehensive sexuality education(CSE); engagement in peer-to-peer education in their village or school on SRH issues; prevention and care of violence against women and girl(VAWG); and counseling on prevention of harmful traditional practice(HTPs) , 11, 22.Adolescent: In this study, adolescents are boys and girls aged 15–19 , 26, 27.Comprehensive sexuality education (CSE): When adolescents were comprehensively offered at least one element of the CSE within the last 12 months from health workers working in any of the service delivery points; awareness creation on reproduction, having information about a positive and respectful approach to sexual relationships, prevention of SRH problems, rights-based approach to SRH services, prevention of violence against women and girls, mental health, and approaches to life skills to enhance their sexual, physical, and emotional wellbeing , 28, 29.Discussion on SRH issues: Adolescents who have discussed at least two SRH issues over the past 12 months  with health care providers, peers, sexual partners, and/or parents .Modern contraceptive service utilization: Adolescents who, over the past 12 months, have used one of the modern methods of contraception , injectables, implants, intrauterine devices, emergency contraceptive pills and spermicide agents) , 26.Accessibility to SRH service: This applies to the perceived distance respondents traveled to reach SRH service delivery points and/or the amount of time they spend. Adolescents residing within a 1.6 km radius of the nearest HRS service center and/or reaching these service delivery points within a 30-min walk were classified as having good geographical access .Substance use: Use of addictive substances such as alcohol, Khat or cigarettes at a frequency of; more repeated than daily, daily, weekly or monthly during the 12 months prior to the study .Reproductive health service knowledge: Adolescents were asked twelve questions about their perceptions of SRH-related issues. An index was created to summarize the level of knowledge, and those who scored higher than the mean were classified as knowledgeable, while those who scored lower were classified as not knowledgeable , 22.Availability of Youth clubs: Accessibility of places/rooms where young people can meet and gather SRH information, SRH services such as contraceptives, physical activities, social support, and peer-to-peer discussion, with the aid of trained workers and volunteers to protect adolescents from negative events, anti-social behavior, crime, drug, and alcohol abuse that are a problem in this community–32.One thousand-nine rural adolescents took part in the study, resulting in a response rate of 98.1%. The average age of adolescents was 17.07 ± 1.4 years and females accounted for more than half (57.1%). The largest proportion of adolescents (95.9%) was single and more than half (51.8%) went to secondary school. Guraghe constituted the majority (90.8%) of the ethnicity and, according to religion, more than half (54.9%) were Orthodox Christians. The average sizes of families were 5.15 ± 1.55. Concerning ease of geographic access, fewer than half (43.3%) of participants had access to these service delivery points within a 30-min walk of their homes. Health centers (60.2%) and private clinics (35.7%) were among the commonly accessed service delivery points. Other service delivery points that adolescents had access to were private pharmacies (32.4%), hospitals (24.5%), and health posts (13.8%). More than four in ten (41.7%) said there were youth clubs (YCs) nearby  had sexual partner/s, of which 62.5% had one sexual partner . Over the past 12 months, 443 (43.9%) study participants reported that they had had a parental discussion on SRH issues. The main topics covered during the discussion were abstinence (60.9%), preventing unwanted pregnancies (77.6%), preventing STIs/HIV/AIDS (62.3%), and contraception (28.4%). In terms of substance use in the previous 12 months, 119 (11.8%), 146 (14.5%), and 103 (10.2%) of respondents used alcohol, Khat, and cigarettes, respectively  of respondents were knowledgeable (i.e. above the mean score of 6.67) about SRH issues. The majority (66.2%) of respondents had information on SRH services and the school environment was the commonest source of information (85.1%). Nearly three-quarters (72.1%) of adolescents knew at least one method of contraception  score of service uptake was 4.05± 1.94), with only 69(6.8%) of adolescents receiving all key elements. Taking into account the different components of care, the delivery of comprehensive sexuality education (CSE) was the commonest received item by adolescents (55.1%), followed by the VCT service (51.0%). While relatively few respondents (25.9%) were provided with contraceptive information and services ] Table .Table 4RThe Sexual and Reproductive Health(SRH) program for adolescents is one of the critical components of health indicators for immediate and long-term SRH needs , 12. ThiIn terms of individual service components, 51.0% of youth received VCT services, which is lower than similar studies elsewhere in Ethiopia , 27. TheIn addition, this research found that on first sexual contact, the use of contraceptives was only 61.7%. This has shown how frequently these adolescents are exposed to multiple sexual and reproductive health problems, such as unwanted pregnancies, unsafe abortions, STIs, and HIV/AIDS. There is also evidence that the content of all other adolescent SRG services is low, implying that stakeholders need a concerted effort to improve these services.The level of education of adolescents was found to influence the use of SRH services. This finding was in tandem with some studies conducted elsewhere , 26, 38.The current study states that discussing with parents has a positive impact on the uptake of SRH services. Some studies carried out in Africa complement this observation , 35, 39.This study found that for adolescents who are not knowledgeable about SRH-related issues, the likelihood of using SRH services is lower than for adolescents who are knowledgeable about SRH-related issues. This is supported by a similar study done in northern Ethiopia . This isAdolescents who had no youth club were 29% less likely to use SRH services than adolescents who reported that there was a youth club in their area. This is consistent with research from African nations –32. The There are strengths as well as limitations in this research. The study is the first of its kind to comprehensively assess the determinants of SRH practice in this area of study and at the country level, taking into consideration all aspects of care. Although efforts have been made to minimize potential gaps in this report, readers should be cautious in their interpretation of the findings. Given that it had some sensitive issues and focused on self-reporting, respondents may have been exposed to social desirability biases and may have contributed to the under-reporting of some SRH services. Finally, there may be a risk of recall bias because adolescents were interviewed about some events that had already occurred before this study.In the study area, the mean score and overall SRH service utilization were low. Educational level, having a parental discussion, lack of youth clubs, and knowledge of SRH issues were identified as significant predictors of the uptake of key elements of SRH services. Schools should be an excellent means of educating adolescents to increase their knowledge of key elements of SRH services. Furthermore, stakeholders must work together to improve the culture of parental discussion with adolescents and establish and strengthen youth clubs, as measures for encouraging the use of SRH services.


High-grade serous ovarian carcinoma (HGSOC) is the most common ovarian cancer type; most patients experience disease recurrence that accumulates chemoresistance, leading to treatment failure. Genomic and transcriptomic features have been associated with differential outcome and treatment response. However, the relationship between events at the gene sequence, copy number, and gene-expression levels remains poorly defined.n = 362) with detailed clinical annotation to interrogate the relationship between patient subgroups defined by specific molecular events.We perform multiomic characterization of a large HGSOC cohort (BRCA2-mutant (BRCA2m) and EMSY-overexpressing cases demonstrated prolonged survival [multivariable hazard ratios (HR) 0.40 and 0.51] and significantly higher first- and second-line chemotherapy response rate. CCNE1-gained (CCNE1g) cases demonstrated underrepresentation of FIGO stage IV cases, with shorter survival but no significant difference in treatment response. We demonstrate marked overlap between the TCGA- and Tothill-derived subtypes. IMR/C2 cases displayed higher BRCA1/2m frequency  and significantly greater immune cell infiltration, whereas PRO/C5 cases had the highest CCNE1g rate  and were uniformly low in immune cell infiltration. The survival benefit for cases with aberrations in homologous recombination repair (HRR) genes was apparent across all transcriptomic subtypes . There was significant co-occurrence of RB loss and HRR gene aberrations; RB loss was further associated with favorable survival within HRR-aberrant cases .These data paint a high-resolution picture of the molecular landscape in HGSOC, better defining patients who may benefit most from specific molecular therapeutics and highlighting those for whom novel treatment strategies are needed to improve outcomes. Molecular features of high-grade serous ovarian carcinoma have been associated with outcome and treatment response. Understanding associations between these different features is important for identifying optimal management strategies for patients. However, the relationship between molecular events at the gene sequence, copy number, and gene-expression levels is poorly understood. We explore these associations and the clinical impact of specific molecular events in a large patient cohort, highlighting patient subgroups with differential survival and therapy sensitivity. We identify significant relationships between subgroups defined by gene-expression patterns, mutation events, copy number events, and levels of infiltrating immune cells. These data suggest specific therapies—such as PARP inhibitors, agents targeting the cell cycle, and immunotherapies—are most likely to benefit particular patient populations. Moreover, loss of RB expression, which frequently occurs alongside homologous recombination defects, may affect patient survival in this context.High-grade serous ovarian carcinoma (HGSOC) is the most common form of tubo-ovarian cancer. The majority of patients with HGSOC are diagnosed at advanced stage and experience poor prognosis, with a five-year survival of approximately 30% in this population . AlthougBRCA1 and BRCA2 (BRCA1/2m) has ultimately paved the way for the integration of poly-(ADP-ribose) polymerase (PARP) inhibitor use into routine care for some patients  diagnosed prior to 2007; available formalin-fixed paraffin-embedded (FFPE) treatment-naïve surgical specimen; first-line platinum-containing chemotherapy; minimum 3-year follow-up. Pathology review of H&E-stained slides was undertaken by expert gynecological pathologists  to identify HGSOC cases ; IHC for p53 and WT1 was used to clarify cases of uncertain histologic type . Ethical approval was obtained from South East Scotland Human Annotated Bioresource (Lothian NRS Bioresource Ethics Committee reference 15/ES/0094-SR705 and SR752). The need for consent was waived by the ethics committee due to the retrospective nature of the study. This study was carried out in accordance with the principles of the Declaration of Helsinki.CCNE1 and EMSY copy number (CN) was quantified by TaqMan qPCR (Supplementary Methods Section 2). Samples with ≥4 copies were considered gained for CCNE1; samples with ≥6 copies of EMSY were considered amplified.H&E-stained slides were marked to identify tumor areas of high cellularity and used as a guide for macrodissection of 10-μm FFPE sections for DNA extraction. DNA extraction was performed using the QIAamp FFPE DNA Kit and Qiagen Deparaffinization Solution (Qiagen). Extracted DNA was quantified by high sensitivity Qubit assay. High-throughput sequencing was performed using a custom Integrated DNA Technologies gene capture panel with unique molecular indices (Supplementary Methods Section 3). Whole-genome libraries were generated, pooled for target capture (see Supplementary Methods Section 3 for a full list of target genes designed around known HGSOC driver events and homologous recombination repair-associated genes), and sequenced using an Illumina NextSeq 550 at the Edinburgh Clinical Research Facility, Western General Hospital, Edinburgh, UK. The median per-sample mean target coverage was 593× . Reads were processed using the bcbio v1.0.6 high-throughput sequence analysis pipeline (Supplementary Methods Section 4). Consensus reads aligned to hg38 underwent variant calling using a majority vote system from three variant callers . Called variants were annotated using the Ensembl VEP v90.9 against Ensembl release 90 and filtered to retain only functional variation (Supplementary Methods Section 5).EMSY-overexpression status (Supplementary Methods Section 6). Briefly, samples from otherwise unselected patients who received adjuvant platinum-based chemotherapy were characterized in this previous work as a training (n = 247 HGSOC) and validation (n = 115 HGSOC) cohort. RNA was extracted from macrodissected FFPE, cDNA was amplified, fragmented, and labeled prior to hybridization to the Ovarian DSA cDNA microarray platform. Raw transcriptomic data were normalized using the Robust Multi-Array Average method prior to a quality control. TCGA  and Tothill  transcriptomic subtyping calls were made with the consensusOv R package using the consesusOv and Helland approaches ; marker-positive cell burden was quantified as percentage positive cells within tumor areas using QuPath version 0.1.2 .PTEN and RB protein loss was detected by IHC using sections of the HGSOC TMA (Supplementary Methods Section 8). Loss was defined as a complete absence of positive staining in tumor cells with confirmed corresponding positive internal control stromal staining.CN analysis was performed using CopywriteR  with 95% confidence intervals (95% CI). For survival analysis adjusted for other clinicopathologic factors, multivariable hazard ratios (mHR) are reported. Median follow-up time was calculated by the reverse Kaplan–Meier method. Adjustment for multiple testing was applied using the Bonferroni method.All statistical analyses were performed using R version 4.0.3 . Comparisons of frequency were performed using the Chi-squared test or Fisher exact test, as appropriate. Between-group comparisons of continuous variables were performed using the Mann–Whitney All data are available upon reasonable request to the corresponding author, subject to requests falling within our local ethics framework. Normalized gene-level transcriptomic data are included as an appendix to this manuscript.n = 27 insufficient tumor, n = 141 non-HGSOC, n = 1 failed sequencing library preparation, n = 8 failed quality control; Supplementary Fig. S1). Clinicopathologic features of the study cohort are summarized in Of the 539 ovarian cancer cases that met eligibility criteria, 362 were classified as HGSOC following pathology review and underwent molecular characterization  demonstrated mutation of other HRR genes . 14.9% of cases displayed CN gain of CCNE1 (CCNE1g) and 6.6% demonstrated amplification of EMSY. Tumors demonstrating EMSY amplification were enriched for EMSY mRNA-overexpressing cases ; however, EMSY CN was a poor predictor of EMSY-overexpression status .The frequency of CCNE1g HRR-wild-type reference population , BRCA2m and EMSY-overexpressing (8.6%) cases demonstrated favorable outcome ; CCNE1g cases demonstrated significantly shorter survival after accounting for age, stage, and debulking status . The BRCA2m and EMSY-overexpressing subgroups demonstrated the highest rates of complete response to first-line chemotherapy ]; however, only BRCA2m and EMSY-overexpressing cases had a significantly higher complete response rate after adjusting for multiple testing . At relapse, BRCA2m and EMSY-overexpressing cases retained a higher chemotherapy response rate . The chemotherapy response rate was similar in the CCNE1g and HRRwt groups at both primary treatment and relapse . There was marked overlap between the subtyping approaches , whereas the C2 subtype demonstrated the lowest CCNE1g frequency  was apparent across all transcriptomic subtypes . We did not observe any significant differences in the overall burden of CN loss or gain events between transcriptional subtypes .Prolonged survival for cases with HRR gene aberrations . BRCA2m cases demonstrated the highest levels of CD3+ infiltration . The IMR/C2 subtypes demonstrated the highest infiltration levels, whereas the PRO/C5 subtypes demonstrated uniformly low levels of infiltrating cells.Subtypes defined by both transcriptomic subgrouping methodologies demonstrated marked differences in infiltrating CD3PTEN CN .10.6% of cases  demonstrated PTEN protein loss ; Fig. 5ARB1 mutation , and there was significant co-occurrence between RB loss and PTEN loss  demonstrated loss of RB protein . In cases with HRR gene aberrations, RB loss was associated with significantly longer survival ; 6 cases were in the EMSY-overexpression HRR-centric subtype (11.3%), and 1 case was in the non-BRCA HRR gene mutant group (1.9%). Three cases had CCNE1g (5.7%), and the remaining 25 were in the HRRwt group (47.2%). Ten percent of cases had PTEN loss  and 29.4% had RB loss . These cases demonstrated a high tumor-infiltrating immune cell burden .Fifty-three patients were alive 5 years following diagnosis of advanced-stage (FIGO III/IV) HGSOC. Ten cases (18.9%) were BRCA1/2m, EMSY overexpression, and non-BRCA HRR gene mutation) demonstrated a higher rate of complete debulking compared with HRRwt cases, but this did not cross the threshold for statistical significance after adjusting for multiple testing . The IMR transcriptomic subtype was associated with significantly higher rates of complete resection (Bonferroni-adjusted P = 0.0134), and complete resection rates were also significantly higher in cases with high CD8+ infiltration .Molecularly defined subgroups were interrogated for association with rates of complete resection. HRR-aberrant cases  and Tothill  subtypes. The MES and PRO TCGA subtypes demonstrated marker overlap with the C1 and C5 Tothill subtypes, whereas the majority of DIF and IMR cases were of the C4 and C2 subtypes, respectively. This overlap is consistent with previous reports of overlap between these subtyping approaches .BRCA1/2m. These cases also demonstrated the highest immune cell infiltration burden, with significantly greater levels of CD3+ and CD8+ cell infiltration. Together, the high BRCA1/2m rate and high levels of immune engagement in IMR/C2 tumors likely underpin the favorable outcome reported in these patient groups  represents a challenge toward identifying further candidate biologically targeted strategies within this patient group. Cyclin E1, the gene product of CCNE1, complexes with CDK2 to drive cell-cycle progression from G1 into S phase  affecting both RB1 and PTEN ; however, this necessitates the use of cases diagnosed prior to implementation of the most contemporary treatment modalities. Specifically, our cohort was not diagnosed in the era of maintenance antiangiogenic or PARPi use for first-line management, and a larger proportion of patients in our cohort underwent single-agent platinum rather than platinum-taxane doublet chemotherapy compared with contemporary cohorts. Moreover, most were treated within the era where achieving <1 cm RD was considered optimal debulking, and the rate of complete surgical resection is lower than that achieved in some modern tertiary centers.We present a large, pathologically confirmed HGSOC patient cohort with extensive follow-up and detailed clinical annotation, including chemotherapy response data. Together with the multiple layers of molecular characterization, these represent major strengths of this work. However, we were unable to characterize genome-wide SVs due to a lack of whole-genome sequencing, which is a limitation of the study. The lack of th HGSOC , and we CCNE1g cases demonstrate poor outcomes despite underrepresentation in the most advanced-stage cases; this suggests inherently aggressive biology rather than a greater propensity for metastatic spread. Previous reports have suggested this may be due to intrinsic chemoresistance  should focus on this patient group in the hope of improving outcomes in this underserved population.Specifically regarding future stratification and rational trial design, our study highlights a number of major points. First, sistance ; howeverEMSY occurs in a group of BRCA1/2 wild-type patients and is associated with a phenotype akin to HRR deficiency . This is consistent with EMSY's known role as a regulator of BRCA2 function  where this may be appropriate, but for many tumors, multiple key events coexist and may independently affect outcome (as is seen for RB status in HRR-deficient patients).Lastly, and more generally, knowledge of potentially targetable molecular events that co-occur or demonstrate mutual exclusivity is crucial to inform future therapeutic strategies. We believe that our study demonstrates that the concept of a simple model where each HGSOC in a patient cohort is ascribed to a single key driver event is outdated. There are some subgroups (e.g., CCNE1g and infiltration of immune cells; integration of these data highlights patient groups which may benefit most from conventional chemotherapy and specific targeted biological agents. Patients with CCNE1g and HRRwt tumors represent those with the greatest unmet clinical need; investigations of new treatment strategies should focus on this patient group. RB and PTEN loss is common in HGSOC and frequently occurs alongside other molecular events, with RB loss affecting a large number of tumors with HRR gene aberrations.Together, these data provide a high-resolution picture of the molecular landscape in HGSOC, integrating genomic sequencing with CN data, transcriptomic profiling, and immune cell infiltration burden in a cohort of HGSOC with rich clinical annotation. Specific transcriptomic subtypes are associated with marked differences in the frequency of HRR gene aberrations, Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.


HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells. Nearly a century has passed since the discovery of HDL and most of HDL research has been focused on intercellular transport of lipids  , 2. AlthThe initial discovery that HDL transport functional microRNAs (miRNA) has led to further profiling studies and the identification of multiple other classes of sRNAs on HDL , 7, 8. FMultiple techniques have been utilized to quantify HDL-sRNAs and evaluate their acquisition from and delivery to cells. Based on quantitative PCR (qPCR), we have previously shown that HDL, complexed with exogenous sRNAs, have the capacity to deliver sRNAs to recipient hepatocytes . On the The study population included N = 25 healthy adult patients . Blood samples were collected following informed written consent from patients and experiments were performed under institutional review board approved protocols from Vanderbilt University Medical Center. These studies abide by the Declaration of Helsinki principles.g for 15 min. Plasma was then frozen and shipped on dry ice to Vanderbilt University Medical Center for processing. Plasma was thawed and filtered through a 0.45 μm syringe filter and its density was adjusted to 1.025 g/ml with KBr. The sample was overlayed with 1.019 g/ml saline buffer, centrifuged at 40,000 R.P.M.  for 24 h at 4°C, and the resultant superior layer  was isolated. The density of the remaining sample was adjusted to 1.080 g/ml with KBr, overlayed with 1.063 g/ml saline, centrifuged, and the resultant superior layer  was collected. The density of the remaining sample was adjusted to 1.225 g/ml with KBr, overlayed with 1.210 g/ml saline, centrifuged, and the resultant superior layer  was collected. Lipoprotein samples were dialyzed into PBS buffer  for four exchanges into 4 L buffer utilizing dialysis tubing .Blood was collected in sodium citrate-containing tubes and plasma was isolated utilizing centrifugation at 1,500 Protein quantification assays were performed by BCA protein assay kit . Total cholesterol quantification assays were performed utilizing cholesterol reagent  and a cholesterol standard (C7509). Triglyceride (TG) quantification assays were performed utilizing TG reagent  and a glycerol standard (G7793). Phospholipid C quantification assays were performed utilizing phospholipid C kit . Assays were performed in a 96-well format according to the manufacturer’s protocol, quantified by a BioTek Synergy Mx microplate reader, and analyzed using the Gen5 software (version 2.01.14).RNA concentration was quantified utilizing an RNA-specific fluorescent dye  and standard curve prepared by serial dilutions of a purified oligonucleotide . Replicates of standards and samples were loaded into a 384-well, 96-well, or 24-well fluorescence plate and background fluorescence was measured (490/530 nm). The dye solution (50 μM) was added to each well to a final concentration of 10 μM and incubated at 37°C for 25 min. The fluorescence of each sample was then measured (490/530 nm), and the background fluorescence was subtracted. The concentrations of the samples were quantified utilizing the single-stranded sRNA standard curve.50.Fluorescence-labelled tRNA-derived sRNA  was denatured and incubated (1:1) with serial dilutions of density-gradient ultracentrifugation (DGUC)-VLDL, DGUC-LDL, or DGUC-HDL in PBS for 5 min and then loaded in glass capillaries (NanoTemper Technologies) and tested on the Monolith NT.115 instrument (NanoTemper Technologies) as previously described . MicroscCellular RNA from J774 macrophages were purified using the Total RNA Purification Kit  according to the manufacturer’s protocol. RNA isolated from lipoproteins, purified by DGUC, was purified by RNeasy Mini Kit  according to the manufacturer’s protocol.Assays were performed using a Take3 Micro-Volume Pate (Biotek) according to the manufacturer’s protocol, quantified with a BioTek Synergy Mx microplate reader, and analyzed using the Gen5 software (version 2.01.14).Assays were performed using the Agilent RNA 6,000 Pico Kit  and processed using a 2,100 Bioanalyzer instrument (Agilent G2939BA), according to the manufacturer’s protocol.RNA digestion was performed by RNase I treatments , according to the manufacturer's protocol. DNA digestion was performed with dsDNase , according to the manufacturer's protocol.3, and pH 7.4). The sample loop was emptied with 5 ml of running buffer prior to a 91.5 ml fractionation period (0.3 ml/min) where 1.5 ml fractions were collected in 96-well plates .Samples were filtered through a 0.2 μm polyvinylidene difluoride centrifugal spin filter  and were brought to a 1 ml total volume with PBS prior to injection into the sample loop of the ÄKTA™ Pure chromatography system (Cytiva). Samples were fractionated utilizing three tandem Superdex® 200 Increase 10/300 Gl columns  in a Tris-HCl buffer  and water ad libitum. Male and female mice ages 8–16 weeks old were used in this study. In all animal experiments described in this study, mice were euthanized by inhalation of 5% isoflurane utilizing the drop jar dosing method and cervical dislocation was performed to ensure the death of the animal.g in a swing bucket rotor for 15 min prior to analyses. Bone marrow cells were isolated from mouse femurs and tibiae, and cultured in treated-cell culture plates for seven days in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher), 1% penicillin/streptomycin (Gibco), L-glutamine , sodium pyruvate (110 mg/L), and murine cytokines GM-CSF (40 ng/ml) or GM-CSF (20 ng/ml) and IL-4 (5 ng/ml) for differentiation into bone marrow derived macrophages or dendritic cells (BMDMs or BMDCs), as previously described (J774A.1 cells (ATCC) were purchased and cultured according to the manufacturer’s protocols. Macrophages were labeled with SYTO RNASelect Green Fluorescent cell stain according to the manufacturer’s protocol. In experiments with HDL treatments, DGUC HDL were added in media to achieve a final protein concentration of 1 mg/ml in DMEM, Thermo Fisher supplemented with 1% penicillin/streptomycin (Gibco), L-glutamine , and sodium pyruvate (110 mg/L). DGUC LDL and transferrin were used as controls for the intercellular RNA transfer assay at final concentration of 0.5 mg/ml in DMEM media. Cell treatments were incubated for 24 h, except RNA efflux experiments, including those with small molecule inhibitors, which were incubated for 4 h. The small molecule inhibitors Block lipid transport-1  and Dynasore (Sigma-Aldrich) were added to media at 10 μM final concentration, 1 h prior to and during RNA export. Scavenger receptor, class B type I (SR-BI) blocking antibodies  were added to the media 1 h prior to and during RNA export experiments at a 1:500 dilution in media, according to the manufacturer’s protocol. Media were isolated from cells and centrifuged at 500 2 throughout imaging. Image panels were prepared with ImageJ software (Version 1.53e) and scale bars lengths represent a 20 nm. Unless specifically outlined, Hoechst 33,342 , LysoTracker Deep Red , and SYTO RNASelect Green Fluorescent cell Stain  were used for live cell imaging in accordance with the manufacturer’s protocol.Macrophages were plated in uncoated 35 mm dishes  at a cell density of 25,000 cells/ml media and grown to approximately 70% confluency prior to imaging. Live cell imaging was performed at 63x resolution using a Zeiss LSM 880 microscope, and analyses were completed with Zen Black software , and samples were maintained at 37°C and at 5% COt-tests were applied. When comparing the means of multiple groups, One-Way ANOVA analyses were performed with Tukey’s post-test to correct for multiple comparisons. To display sample dispersion, values were reported as either the sample mean ± standard deviations or violin plots showing median and quartile ranges. A P-values <0.05 were considered statistically significant. Graph generation and statistical analyses were performed utilizing the Graphpad Prism 9 software.When comparing between two independent groups, unpaired two-tailed The most common methods of assessing extracellular sRNA levels in biofluids involve RNA isolation steps, including solvents and silica frit-based columns, which likely have inherent biases. To quantify total RNA levels in isolated RNA samples, spectrophotometry and/or electrophoresis approaches are normally completed; however, these are time-consuming and lack sensitivity. Here, chloroform and ethanol extraction of RNA with silica frit-containing columns were used to isolate RNA from human lipoproteins, and total RNA content was assessed by bioanalyzer analysis (Agilent) A. These P > 0.1) between their calculations of isolated macrophage RNA  was observed between the total cholesterol content on HDL with its RNA cargo  and LDL   observed between PC and RNA in VLDL  between TG and RNA in LDL , exomeres, and supermeres , 14, 15.proteins . Resultsproteins D. These proteins D. To assproteins . A signiNA cargo E. This c= 0.271) F, G. The= 0.271) . There w in VLDL  and a siA in LDL . ResultsSome classes of EVs have been reported to copurify with lipoproteins when using density-based isolation methods. Therefore, we sought to assess potential EV contamination in our DGUC-purified lipoprotein samples . We firsP < 0.0001) when HDL was present in the accepting media, as 158.30 ng of cellular RNA / μg cellular protein were quantified in the HDL-containing media (P < 0.0001) in RNA export to HDL in macrophages (P < 0.01) when cells were given HDL derived from FH patients as an acceptor of RNA export, as 161.30 ng of cellular RNA / μg cellular protein was quantified in the HDL-containing media  but statistically increased compared to both the SYTO-only (P < 0.001) and transferrin-supplemented (P < 0.001) conditions  , 30, 31.Results from SYTO analyses suggest that both HDL and LDL transport μg quantities of RNA per mg of total protein. This level of RNA cargo has the potential to activate RNA-sensing toll-like receptors (TLR) within the innate immune system if delivered to specific immune cells. As shown by live cell imaging, our data supports the hypothesis that HDL-delivered RNAs are taken up into the endo-lysosomal compartment of recipient macrophages where they could play a role in TLR signaling. The approaches outlined in this study would enable future studies to characterize the pathway of HDL or LDL-delivered sRNAs through the endo-lysosomal system, characterize the modulators of this process, and colocalize these RNA cargos with TLRs that drive inflammatory pathways. As indicated by previous reports of proinflammatory TLR signaling derived from LDL-associated RNA, it may be possible that HDL’s anti-inflammatory role is mediated, in part, by inducing the efflux of LDL deposited proinflammatory sRNAs within the endo-lysosomal system . HistoriPrevious studies have characterized the pathways involved in HDL-mediated export of host-derived miRNAs from beta cells; however, the pathways by which lipoproteins obtain nonhost sRNAs are poorly understood . The chaAlthough the approaches outlined in this paper offer clear advantages in characterizing and quantifying lipoprotein-mediated RNA transport over conventional tools, it has its own limitations and should be utilized with orthogonal methods that offer sequence identification and epitranscriptomic analysis of the RNA cargo. For example, the use of SYTO RNA labeling approaches provides researchers with a powerful tool to assess global sRNA flux related to lipoproteins that can be followed by sequencing or PCR studies to evaluate the individual identification, cellular origin, and potential biological functions of candidate sRNAs. To summarize, this study a) provides isolation-free wholistic quantification of lipoprotein-associated RNA cargos, b) characterizes the ability of HDL to accept and deliver RNA cargos to cells, c) demonstrates the impact of disease (FH) on HDL-mediated RNA efflux, and d) defines the ability of HDL to serve as a mediator of cellular RNA transfer between immune cells. This study indicates that the sheer mass of RNA cargos carried by lipoproteins, accepted from cells, and delivered to recipient cells by HDL has been unappreciated utilizing previously described methods and offers critical insights to the field of extracellular RNA transport.Data that support the findings of this study are available from the corresponding author, KCV, upon reasonable request.This article contains No author reports a financial interest with respect to the work outlined in this document.


Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m Current intense research efforts promote a coalescence of RNA modification data into a more complete picture of what is now known as ‘the epitranscriptome’. Our knowledge concerning chemical structure, position, stoichiometry and dynamics of eukaryotic modifications in general, and of mammalian ones in particular, have entered the system-wide stage. However, in as much as the term epitranscriptome includes all post-transcriptional events, its understanding must include the arguably most widespread chemical manipulation of RNA, that is: cleavage. Hydrolysis of RNA phosphoester bonds commences directly after transcription and shapes the (epi)trancriptome all the way to nucleoside excretion or salvage, including processing steps like splicing, trimming and RNA degradation in the exosome . Artificin cellulo versus in vitro, i.e. during the isolation procedure, are entirely unclear. Moving further along these lines, small RNAs are also subject to eventual turnover, and thus vanish from the small RNA fraction along with their modifications , we decided to trace the fate of rRNA and tRNAs during degradation via detection of their characteristic modifications. We found that, in line with large sections of published literature, certain modifications or combinations thereof, are unique or sufficiently characteristic to a given RNA type, and allow tracing of corresponding RNA species in the course of continuous degradation to ever shorter RNA fragments. In particular, modifications typically found in tRNAs and rRNAs emerge in the small RNA fraction as a consequence of continued degradation. Pronounced differences were found among different tissues, in particular between liver and brain. As a major finding, we define conditions under which characteristic modification patterns for the distinction among different brain tissues become apparent.We therefore perceive an urgent requirement for a characterization of degradation events in tissues after organismal death, in order to determine conditions under which RNA isolates would reflect the physiological state, unbiased by artificial degradation. Because of the known problems with RNAseq (C57BL/6 mice for Mainz based experiments were purchased from Janvier Labs. B6.129S mice were provided by JAX (MMRRC). After delivery, mice were kept for at least 14 days in the animal facility to reduce stress associated with transport. Animals were housed in groups in standard cages. They were provided with food and water ad libitum, with paper towels as cage enrichment, and kept on a 12:12 h light:dark cycle (lights on at 0700 hours). Animals were killed by dissection according to European Communities Council Directive (86/609/EEC) and approved by the local governmental commission for animal health. Heidelberg based experiments were conducted on C57Bl/6J mice at the German Cancer Research Centre (DKFZ) according to applicable laws and regulations. Animal experiments were conducted on adult mice at age between 5–8-month-old.®  for 100 mg tissue, followed by addition of 200 μl chloroform . After incubation (10 min) at room temperature (RT), tissue suspension was centrifuged . The upper aqueous phase was taken and mixed in a 1:1 ratio with isopropanol  and 1 μl glycogen  was added, incubated at RT (5 min) and centrifuged . The RNA pellet was washed twice with ice-cold 75% ethanol including centrifugation . RNA was reconstituted in 50 μl nuclease-free water . RNA concentrations were determined using UV-VIS spectrophotometer Nanodrop 2000  and the integrity of the RNA was checked using the Agilent TapeStation 4200 system  analysis to obtain eRIN values.Mice were sacrificed using cervical dislocation. Brain areas, whole brain (without olfactory bulb and brainstem) and liver tissue were dissected. Total RNA was prepared using 1 ml TRI Reagent® Sequencing gel concentrate , Rotiphorese® Sequencing gel diluent  and Rotiphorese® Sequencing gel buffer concentrate . The dimensions of the gels were 35 cm × 20 cm × 0.01 cm (H × B × T) and seven pockets with 2 cm width. 35 μg total RNA was mixed with 6× TriTrack DNA loading dye  in a 1:3 mixture; the loaded volume did not extent 50 μl per pocket. Gels were pre-run for 30 min limited by the current to 40 mA and the power to 300 V using the Bio-Rad PowerPac™ 300 . The gel running apparatus BRL Model SA was provided by Life Technologies Inc. . After harvesting the gels, they were stained in aqueous ethidium bromide solution  for 5 min and scanned on a Fusion Pulse TS  with 400 ms exposure time. In order to recieve tRNAs a band between 60 and 75 nucleotides (nt) was dissected according to the ultra-low range size ladder . Second, a rather broad band corresponding to ∼15–50 nt was dissected and named RNA fragment (RF) fraction. RNA was eluted from the gel pieces using the ZR small-RNA™ PAGE Recovery Kit for 20 preparations  according to the manufacturers specifications. RNA Recovery Buffer (R1070-1), RNA MAX Buffer (R1070-2), RNA Prep Buffer (R1060-2), RNA Wash Buffer (R1003-3), Zymo-Spin™ IC Columns (C1004), Zymo-Spin™ IIICG Columns (C1006), Zymo-Spin™ IV Columns (C1007), Collection Tubes (C1001), DNase/RNase-Free Water (W1001). In the last step, RNA was redissolved in 20 μl of the nuclease free water . The RNA concentration was again determined using the UV-VIS spectrophotometer Nanodrop 2000.Total RNA was further separated for tRNA and RNA fragments (RF) by 10% urea polyacrylamide gel electrophoresis (PAGE) for at least 1 h. The gel solution was mixed using a 50:40:10 ratio of Rotiphorese®Gold  was cast using two combs, with comb #1 conventionally placed near the cathode and comb #2 placed towards the anode end of the gel. The RNA sample in 1× loading dye containing formamide  was applied to the wells left by comb #1. Progress of electrophoresis was monitored in real-time by a Blue Light Transilluminator . The electrophoresis was performed at 180 V for about 90 min until the first fraction of interest (rRNA) reached the pockets resulting from comb #2. The respective fractions were removed during their passage through the second pocket by pipetting, and precipitated after addition of ammonium acetate/ethanol , 1 μl glycogen  and 2 vol. 100% ethanol ). The samples were incubated at −80°C for 1 h or at −20°C overnight. The RNA pellet was collected after centrifugation at 12 000 g at −4°C for 45 min, washed with 75% ethanol and again centrifuged at 12 000 g at −4°C for 15 min. The resulting RNA pellet was dissolved in ultrapure water. All samples were filtered through 0.2 μm solid phase filters .A prestained 0.8% agarose gel  containing 1× TBE and 1× SYBRPurification from total RNA was performed according to . In brieP. citrinum (Sigma-Aldrich), 0.2 U snake venom phosphodiesterase from C. adamanteus (Worthington), 2 U FastAP (Thermo Scientific), 10 U Benzonase (Sigma-Aldrich), 200 ng Pentostatin (Sigma-Aldrich) and 25 mM ammonium acetate  over night at 37°C in a total volume of 30 μl. For each technical replicate (injection to the LC−MS/MS system) 150 ng of tRNA was digested. For RF-RNA at minimum 75 ng was used per injection.Up to 300 ng of tRNA or RF-RNA per sample was digested to nucleosides using 0.6 U nuclease P1 from 13C internal standard and the sample was prepared. The internal standard dilution (ISTD) is a long-lasting stock mix of 50 ng/μl Escherichia coli and 50 ng/μl Saccharomyces cerevisiae. 1.1 μl ISTD was added per injection to the 30 μl volume accounting for the 10% of sample, that cannot be taken by the syringe of the auto sampler. All measurements were performed on an 1260 Infinity II LC  coupled to an Agilent 6460 Triple Quadrupole mass detection system . The separation was conducted on a Synergi™ Fusion RP C18 column  from Phenomenex . Column oven was set to 35°C. Moreover a gradient method termed NUCS4 was used, defined by the following conditions. Phase A containing acetonitrile (ACN)  starting at 0% and increased in 10 min to 8% ACN. Phase B contained NH4OAc buffer (pH 5.3). This was followed by a steeper gradient to 40% ACN in 10 min. Equilibration time was another 10 min. The flow rate was 0.35 ml/min. 5 mM NH4OAc buffer was freshly prepared. More hydrophobic modifications were not included. The UV trace was recorded in a Multiple wavelength detector (MWD) detector at 254 nm at an attenuation of 1000 mAU at a sample rate of 2.5 Hz.For the sample injection a mixture of the 13C-labeled counterparts: Am, Cm, D, Gm, I, i6A, m1A, m1G, m2,2G, m6,6A, m2A, m2G, m3C, m3U, m5C, m5U, m6A, m7G, mcm5U, ms2i6A, Psi, Q, S2C, S2U, t6A, Tm, Um (abbreviations according to MODOMICS). Parameters of the acquisition method are given in Table The sample was injected to the Mass Spectrometer via the EJS ESI Source in the positive ion mode. The following parameters were defined for the measurement: Capillary current 5400 nA, gas temperature 350°C, sheath gas temperature 345°C, sheath gas flow 10.0 l/min, gas flow 8 l/min, Nebulizer 50.0 psi, Corona voltage 0 V. MRMs were programmed with a window of 6 min around the optimized elution time for the following modifications and their 13C SILIS  were extracted in Skyline 20.1.0.76  and allowed integration of respective peaks, providing values for the area under the curve (AUC). Only peaks with a normal distributed peak shape and a minimum signal-to-noise ratio of 5 in at least one of several compared samples were scored in further analysis (heatmaps or PCAs), while modifications with low abundance in all compared samples were excluded (shown in grey below heatmaps). Those modifications contained in the eviously .k-means value and accounting for a maximum of 300 clusters. One- or two-sided clusters were used alternatively. Principle Component Analysis was performed with five components, and a P-value threshold of 0.05. In cases with ≥3 replicates two-sided Student's t-tests were performed with a P-value of 0.01 to account for the low number of parameters/modifications.Statistical procedures were applied in Perseus 1.509 Software released . Data was normalized by dividing each row by its mean for cluster analysis driven heatmaps generation using the One male mouse (B6.129) was sacrificed using cervical dislocation at the age of 12 weeks. Whole brain (without olfactory bulb and brainstem) and liver tissue was dissected and placed in ice cold phosphate buffered saline (PBS) to rinse away blood.In the first purification step, tissue was cut with a scalpel and then transferred into the homogenization vessel with 6 ml of Mitochondria Isolation Buffer. Homogenization was performed in a Braun Potter S Homogenizor  on ice and five passes were executed. Homogenates were transferred into a new tube and centrifuged with a fixed angle rotor . The supernatant was saved in a new tube and placed on ice, while the pellet was re-suspended in 500 μl MIB and centrifuged again . Both supernatants were combined before the high-spin centrifugation step . Supernatant was discarded and the mitochondria pellet was resuspended in 500 μl MIB.® gradient . For this purpose, liver and brain mitochondria obtained from the first part of the protocol were resuspended in 28 ml of a 12%-Percoll® dilution in MIB. This solution was then carefully applied to a two-step gradient with 7 ml 26%-Percoll® in the upper layer and 4 ml 40%-Percoll® in the bottom layer. For best resolution, the 28 ml brain and the 28 mL liver mitochondria were divided onto four gradient tubes each. A first ultracentrifugation step was performed in a fixed angle rotor  in a Beckman Optima™ LE-80K centrifuge  . Afterwards lower intermediate layer was removed with a SOFT-JECT® syringe  and 17G Hamilton™ needles . The obtained mitochondrial extracts were united in a new tube and filled with MIB up to 20 ml. After second ultracentrifugation  mitochondria were accumulated in a fluffy pellet. In the last step, the pellet was divided into two tubes, one for RNA-isolation and one for western blot analysis. For isolation of mitochondrial RNA, the pellet was dissolved in 800 μl TRI Reagent® and mixed with 200 μl chloroform. Further steps were performed as described above. Pellet was reconstituted in 12 μl nuclease-free water.For obtaining higher purity, mitochondria were further enriched and concentrated in a discontinuous Percoll® 20 , ad 1 l water), membranes were treated with horseradish peroxide conjugated secondary antibodies. For TOMM20 an anti-rabbit antibody was used  and for alpha-tubulin an anti-mouse-antibody was used . In the last step, membranes were washed again three times with 0.5% TBST and analyzed with Amersham ECL™ Prime Western Blotting Reagent  in Fusion Pulse TS.To assess quality of mitochondrial extracts, half of the obtained pellet was dissolved in 200 μl lysis buffer containing 50 mM Tris–HCl , 150 mM sodium chloride , 1% Triton™ X-100 , 0.5% sodium deoxycholate , 0.1% sodium dodecyl sulfate , 5 mM ethylenediaminetetraacetic acid  and 1mM phenylmethylsulfonylfluorid . After incubation on ice for 45 min and centrifugation , proteins were located in the supernatant. After protein determination (Bradford method), 20 μg protein per lane were loaded on a 12% SDS-PAGE and separated by electrophoresis. Samples were transferred onto a polyvinylidene fluoride membrane , incubated for 1 h with 5% Bovine Serum Albumin (BSA)  blocking solution at RT and then incubated with primary antibodies for TOMM20  and alpha-tubulin  for 1 h at RT. After washing three times with 0.5% TBST , sodium chloride 150 mM, 0.5% Tween4Ac, pH 7.5) and degradation was started by addition of 105 μl RNAse T1/A mix . Aliquots  were taken at various time points and added to an extraction mix consisting of 200 μl nuclease-free water, 150 μl phenol  and 150 μl chloroform. After agitation for 30 s, phases were separated by centrifugation . The upper aqueous phase was re-extracted with 100 μl chloroform. The aqueous phase was supplemented with 0.5 μl glycogen and incubated with 2-propanol for 15 min at RT. After centrifugation for 30 min , the supernatant was removed and the pellet with washed with 75% ice-cold ethanol. The supernatant was discarded, and the pellet was air-dried for about 5 min. The degradation status was monitored on Agilent 4200 TapeStation system according to the manufacturers instructions. Digested RNA of each time point was separated by a 10% urea PAGE, and tRNA and RF fractions were excised as describedand processed for mass spectrometry analysis.210 μg of total RNA prepared from fresh mouse liver in a volume of 435 μl water were supplemented with 60 μl of 10x digestion buffer  were sacrificed at the same time by means of cervical dislocation. Mice were at the age of 5–6 months and in total 10 male and 10 female mice were used. Whole brain and liver pieces of the left lobe (∼200 mg) were dissected after different storage times  at RT, to monitor 6,6A in tRNA fractions, were considered degraded and excluded from further analysis according to the rationale developed in the results and discussion section.Cohort 1, from Mainz, Germany: Female and male C57BL/6JRj mice were sacrificed at the age of 12 weeks on the same day. Hippocampus, cortex and cerebellum were dissected manually. For the sake of sufficient RNA input amount, brain areas of two individuals were pooled resulting in four biological replicates. All biological replicates were submitted to a 10% urea PAGE and tRNA- and RF-RNA fractions were isolated and analyzed for their modification content as described above. Biological samples with high levels of ribosomal markers Am and mAt the onset of our investigations, we posited that modifications, properly quantified in cellular RNA, should be informative of several features of the state of a tissue or cell culture, potentially including aspects of cell death, protein synthesis capacity, and mitochondrial biomass or activity . As an eBased on literature on the occurrence of modifications in different RNA species ,40, we pThe substantial body of knowledge on the modification content of eukaryotic tRNA, rRNA and mRNA in the literature firmly establishes that, despite similarities, each species, tissue, and even physiological state features a specifically adapted epitranscriptome ,41,42. Fvide supra. Total RNA (labelled ‘input’) clusters with the rRNA, plausibly as a consequence of the large mass contribution (>80%) of rRNA to cellular RNA populations.Using a stable isotope labelled internal standard (SILIS)  the modi3U, Nm and m6,6A on one hand, and typical tRNA modifications on the other hand.The characteristic patterns of modification content in different RNA populations apparent from the heatmap in Figure 6,6A, which occurs almost exclusively in the small ribosomal subunit 18S rRNA. m3U as counterpart, is typical for the large subunit 28S rRNA , but all of them in lesser amounts than in other RNA fractions, as can be seen in the heatmap of Figure 6A. The presence of m6,6A corroborates small amounts of rRNA contaminations, which were already anticipated from a peak of residual rRNA in the electropherogram of the purified polyA fraction  which is shown in Figure 6,6A and Am truly stand characteristically separated from the rest, presumably because they are contained in rRNA fragments of similar size, undergoing similar degradation events. Their covariation effectively validates these two modifications as small subunit rRNA markers according to our hypothesis (ii). Among the smaller RNAs in the RF fraction, however, there is no more coherent behaviour among the rRNA modifications; a plausible explanation would be that they enter the tRNA fraction in fragments of similar size and metabolic stability, while these properties would diverge upon further degradation.For experimental verification, RNA samples from a degradation experiment were size fractionated by PAGE, excised from gel slabs, eluted, precipitated, and analysed for their modification content by LC–MS as before . The resIn summary, the progressive degradation causes two opposing trends, namely an enrichment of rRNA modifications in the tRNA fraction, and in the same fraction, a dilution of tRNA modifications. The latter simultaneously become enriched in the RF fraction, effectively validating out hypothesis (iii).2i6A, a lipophilic tRNA hypermodification of prokaryotic origin, that is known to occur in mammalian mitochondria, but not in the cytosol or nucleus ), brain mitochondria contain high amounts of queuosine, a wobble modification directly involved in the mRNA decoding process.Of some interest is the detection of ms nucleus . In the  nucleus . Cytosolin vitro degradation as a potential source of rRNA fragments in small RNA preparations, we turned to more physiologically plausible origins of degraded rRNA, such as necrotic or apoptotic processes. These cause a certain amount of dead cells among cells in tissue as well as in tissue culture, and the resulting dead cells can reasonably be expected to contribute RNA to total RNA preparations. Given that degradation processes commence during cell death already, it can furthermore be assumed to follow at least partially predefined pathways. Consequently, the resulting degradation patterns would likely be different from that caused by the artificial in vitro settings above.Having characterized in cellulo between brain and liver tissue after organismal death. Mouse brain and liver were removed at defined time points of 0, 1, 8 and 24 h after death . Degradation in liver was fast progressing in comparison to brain, where even after 24 h, total RNA was still mostly intact. The accelerated degradation in liver relative to brain is easily appreciated in a plot of integrity values vs. time —vide supra.First, two different isolation procedures were tested to gauge the influence of flash freezing and thawing on degradation. In one case, tissue was flash frozen in liquid nitrogen and later minced in RNA isolation solution e Figure . A compan Figure . At 24 hvia heatmaps, as shown in Figure in vitro degradation  were increasing in the tRNA fraction with increasing degradation, while tRNA modifications diminished  allows a clear distinction of all three tissues via unsupervised hierarchical clustering. As before, there is also a distinction of tRNAs versus RNA fragments, but only the tRNA profiles are informative with respect to tissue distinction. Of interest, tissue distinction within the tRNA fraction is also evident in a 3D PCA analysis . Figure s Figure . For verPost mortem RNA degradation, though a plausible source of RNA artefacts, has remained largely ignored as a source of fragmented small RNAs in both, RNAseq and LC-MS based investigations in epitranscriptomics. By analysing decay processes after organismal death, we here established a baseline to gauge the minimum occurrence of rRNA degradation encountered in RNA preparations from fresh tissue. A number of important conclusions flow from these results, pertaining to the identification and validated use of marker modifications, their size distribution upon RNA degradation, and the application to RNA from different tissues.6,6A and Am in the tRNA fraction, but not in the RF fraction, in vitro as well as in cellulo, and in liver as well as in brain. A plausible explanation here must assume that this fraction is, from the very onset, in a steady-state, where the amount of e.g. m6,6A/Am containing fragments being degraded equals that of newly generated fragments. In contrast, the RNA fraction of tRNA size does accumulate rRNA fragments in all settings investigated, meaning that e.g. the m6,6A/Am content in the tRNA fraction can serve as a marker of the overall degradation state of a given sample.Degradation resulted in a clear increase of the co-varying rRNA modifications min vitro degradation and in cellulo degradation in liver, but not in brain, the latter being remarkably inert to degradation altogether. Of note tRNA degradation alone can not per se explain this observation, since there must be an increase of RNA fragments of tRNA size that do not contain tRNA modifications. Since a massive surge in de novo transcription of tRNA is implausible, the decrease of tRNA modification content in an RNA fraction of tRNA size is best explained by a dilution of this fraction with rRNA fragments of corresponding size, which do not carry the respective tRNA marker modifications. Initially, the decrease of typical tRNA modifications in the tRNA fraction is paralleled by an increase in the FR fraction . In brain, degradation is very slow and especially changes in tRNA modifications are much less pronounced in the tRNA fraction, whereas they do increase in the RF fraction at later time points . Of note, these differences do not rely on the abundance of single modifications, which by themselves could be assessed via a simple t Figure , but the and S5. via modification markers, our analysis demonstrates variations in the tRNA epitranscriptome of different brain tissues. Pending a similar assessment of e.g. rRNA modifications, we predict that similar analysis will allow a distinction between many more different tissues, paving the way to a fast characterization of a significant part of the respective epitranscriptomes.Together, these results prompt us to conclude that, based on the prior elaboration of degradation status 6,6A rRNA marker modification, whose whereabouts we have traced in here in detail. The presence of m6,6A in was detected in ‘small’ RNAs by LC–MS (6,6A-containing small RNAs with biological function, whose sequences were, however, not analyzed. Here, our findings point out an important, but by now overlooked fact: there are indeed debris in the small RNA fraction, and some of them are just that: degradation products on their way to recycling.As outlined, the early days of small RNA research have seen a challenge of the ‘debris’ at the bottom of PAGE gels . Numerouby LC–MS  and its A selected list of abbreviations is available in the Supplementary Data.gkab1150_Supplemental_FileClick here for additional data file.


Through idealized, numerical models this paper investigates flows on a reef geometry which has received significant attention in the literature; a shallow, fringing reef with deeper, shore-ward pools or lagoons. Given identical model geometries and varying only reef flat drag coefficients between model runs ( Reference3 suggest that the two end members of this 1D balance are the ‘open reef’, described by balancing an onshore pressure gradient force by bed friction, and the ‘closed reef’, in which an onshore radiation stress gradient is balanced by an offshore pressure gradient force. The 1D model gives a satisfactory description of some observed flows (e.g.5), however, the horizontal velocity profile is unaccounted for, and some observations reveal vertically sheared flows similar to beach undertow that cannot be captured by the existing model9. While a deviation from the established 1D model is both expected and documented, so far no framework has been presented to interpret this departure and its implications for reef-wide circulation.The present study investigates the effect of varying reef roughness on dynamics and residence times on an idealized reef. Wave-driven flows on coral reefs are typically described by a one-dimensional (1D), depth-averaged model, which details the approach and breaking of waves on a shallow fore-reef or reef crest, and estimates the onshore or cross-reef flow (see e.g.11) where the cross-shore mass flux is zero. However, reef systems often exhibit alongshore bathymetric variability allowing for a nonzero flux, the effect of which was studied by Ref.12, who showed through numerical simulations that an increased onshore mass flux was correlated with a decrease in undertow. Reference6 observed in experiments and simulations that undertow decreased as roughness increased, and Refs.9 observed that reef undertow coincided with high water levels, and ceased during low tide.Undertow has been observed and described as it manifests on sandy beaches , and that the associated circulation results in markedly longer residence times. “With Ref.x) direction is given by13h is the depth, u is the depth-averaged, Lagrangian velocity, The 1D depth-integrated, wave-averaged momentum equation on a shallow reef flat in the cross-reef , the radiation stress gradient (RSG), and the bottom friction (BF) terms. In Ref.14z is the vertical coordinate, 15), and the displacement height d, which we will neglect for simplicity. Thus, in the absence of a significant RSG in Eq.  relative to the reef flat is 0.6 m and the tidal amplitude is 0.5 m, defined at the offshore boundary. All other boundaries are closed. Additionally, the offshore boundary is forced by a JONSWAP spectrum with 31. Additionally, the fringing reef is a commonly occurring geometry, see e.g., Ningaloo Reef33, Oahu34, Guam35, Faga’alu Bay36, and many others.The geometry and forcing of the numerical model roughly represents the southern reefs of Ofu, American Samoa, which have been studied in detail by Refs.38 found on coral reefs. Following e.g.39, the velocity field is seeded with non-inertial tracer particles at a time Particle tracking is calculated in post processing using the simulated 3D velocity fields and vertical eddy viscosity of the w is the vertical velocity and Horizontally, the position at time step 39 studied a numerical model of the enclosed Mururoa lagoon, French Polynesia, in which an atoll and distinct passages defined the lagoon relative to the surrounding ocean. This geometry lends itself well to bulk estimates such as turnover time 40 where V is the lagoon volume and Q the net flow defined at the passages. In comparison, Ref.36 studied Faga’alu Bay, American Samoa, where the demarcation between the reef inflow and outflow was less pronounced. Using Lagrangian drifters, they defined residence time by the velocity of a drifter and the distance traveled, normalized by a representative area over which the reef had similar characteristics, thereby estimating spatial residence times of different patches of the reef.Reef geometry varies significantly between sites, and thus residence times must be calculated with care. For example, Ref.40. The reef domain is seeded with particles, and particle trajectories are calculated with the advection–diffusion Eq. . Particles are released an hour before low tide and tracked for four hours, and the residence times are found in Fig. Here, particles are initiated in all points across the reef flat, pools, and channels, as apposed to the reef flat only. Particles are tracked, and the time at which a particle leaves the system is noted  with residence times generally less than 2 h.Results from idealized numerical models presented in this paper show how varying reef flat roughness on a reef can substantially change the circulation and thus the residence time on the reef. Models with high reef roughness and shallow reefs follow a classic reef circulation dynamic, with unidirectional, onshore reef flow that passes through deeper pools and channels alongshore behind the reef flat, and exits through channels transecting the reef flat. On the reef flat, an onshore pressure gradient is balanced by an offshore bed shear stress, and the flow is similar to open channel flowModels with low reef roughness and deeper reefs are characterized by sheared cross-reef flow, at times with a distinct offshore flow on the reef flat similar to beach undertow. On this reef flat, radiation stresses and barotropic pressure balance, referred to here as a closed (beach-like) reef. In spite of a strong offshore flow along the reef flat these models exhibit much longer residence times. This is in part due to strong vertical mixing offshore of the reef due to wave breaking which allows for recirculation, as well as entrapment in two counter-rotating horizontal eddies in the shoreward pools. Thus, the undertow is not effective at flushing the reef system and pools. Residence times for these models approached 5 h, not including the channels which consistently have a low residence time.21). For these reefs, this study identifies a potential positive feedback mechanism where wide-spread bleaching events and coral death decreases reef roughness by lowering the geometric complexity of the sea bed, which in turn can transition a reef system from efficient flushing and short residence times to inefficient flushing and long residence times. Corals depend on their physical environment to bring in nutrients and cool offshore waters, and a less efficient circulation pattern could lead to further coral decline. Additionally, the tidal dependency found in this study shows that reefs that are currently open and efficiently flushing could transition to a closed, inefficiently flushing circulation pattern given sea level rise.The fringing reef type studied here is representative of reefs across the globe and has been studied extensively previously (e.g.Supplementary Figure 1.Supplementary Figure 2.Supplementary Video 1.Supplementary Video 2.Supplementary Legends.