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Background: Intensive care unit (ICU) is a multidisciplinary staffed and specially equipped area of a hospital dedicated to providing quality care for patients with life-threatening conditions. The provision of intensive care services to critically ill patients is a global issue. Though care is advancing globally, the quality of available ICUs in Ethiopia is still lagging far from the standard. Objective: This study aimed to assess ICU mortality and its associated factors among patients at Tibebe Ghion specialized hospital, Northwest Ethiopia, 2021. Method: A retrospective cohort study was conducted from February 24th, 2019 to January 24th, 2021. Data were collected from medical records by using pretested structured data retrieval checklist. The collected data was entered into Epi-data version 3.1 and analyzed using R version 4.0 software. Descriptive statistics computed. A simple logistic analysis was run (at 95% CI and p-value < 0.05) to identify the determinants for ICU mortality. Results: A total of 568 study participants’ charts were reviewed. The median length of ICU stay was 4 days. The overall mortality rate of the ICU-admitted patients was 29.6% (95% CI: 26%, 33%). Admitted in 2020 (AOR=0.51; 95%CI: 0.31, 0.85), having altered mentation (AOR=13.44; 95%CI: 5.77, 31.27), mechanical ventilator used (AOR = 4.11; 95%CI: 2.63, 6.43), and stayed < 5 days in the ICU (AOR = 3.74; 95%CI: 2.31, 6.06) were significantly associated with ICU mortality. Conclusion: The ICU mortality rate was 29.6%. Years of admission, altered mentation, mechanical ventilator use and days of stay in the ICU were the predictors for ICU mortality. Improvement efforts led by the Ministry of Health with input from the acute care workforce are an urgent priority.

RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

The origin and contribution of seminal plasma RNAs into the whole semen RNA repertoire are poorly known, frequently being overlooked or neglected. In this study, we used high-throughput sequencing and RT-qPCR to profile microRNA (miRNA) constituents in the whole semen, as well as in fractionated spermatozoa and seminal plasma of Atlantic salmon (Salmo salar). We found 85 differentially accumulated miRNAs between spermatozoa and the seminal plasma. We identified a number of seminal plasma-enriched and spermatozoa-enriched miRNAs. We localized the expression of some miRNAs in juvenile and mature testes. Two abundant miRNAs, miR-92a-3p and miR-202-5p, localized to both spermatogonia and somatic supporting cells in immature testis, and they were also highly abundant in somatic cells in mature testis. miR-15c-5p, miR-30d-5p, miR-93a-5p, and miR-730-5p were detected only in mature testis. miRs 92a-3p, 202-5p, 15c-5p, and 30d-5p were also detected in a juvenile ovary. The RT-qPCR experiment demonstrated lack of correlation in miRNA transcript levels in seminal plasma versus blood plasma. Our results indicate that salmon semen is rich in miRNAs, which are present in both spermatozoa and seminal plasma. Testicular-supporting somatic cells are likely the source of seminal plasma enrichment, whereas blood plasma is unlikely to contribute to the seminal plasma miRNA repertoire.

Summary: Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5′ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step. : Giess et al. introduce RCP-seq, a method to study small ribosomal subunit (SSU) dynamics in zebrafish. It reveals threading as the main mode of recruitment to the mRNA and quantifies the impact of sequence features that affect SSU processivity. By contrasting scanning to translating ribosomes, the study calculates initiation rates and redefines the optimal translation initiation context for zebrafish. Keywords: translation, RNA, ribosome profiling, next-generation sequencing, small ribosomal subunit (SSU), scanning processivity, initiation, ribosome recruitment, uORF, Kozak sequence

Translation initiation is often attributed as the rate determining step of eukaryotic protein synthesis and key to gene expression control1. Despite this centrality the series of steps involved in this process are poorly understood2,3. Here we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5’ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features regulating the recruitment and processivity of scanning ribosomes, redefine optimal initiation contexts and demonstrate endoplasmic reticulum specific regulation of initiation. Our approach enables deconvoluting translation initiation into separate stages and identifying the regulators at each step.

Biological functions of micro RNAs (miRNAs) in the early stages of vertebrate development remain largely unknown. In zebrafish, miRNA miR-92a-3p is abundant in the germ cells throughout gonadal development, as well as in ovulated oocytes. Previously, we demonstrated that inhibition of miR-92a-3p in mature ovaries resulted in developmental arrest at the 1-cell stage upon fertilization of the affected oocytes. This suggested functions of miR-92a-3p in early development. In the present study, we identified wee2, an oocyte-specific protein tyrosine kinase, as a target of maternal miR-92a-3p during the early stages of zebrafish embryogenesis. Spatiotemporal co-presence of both miR-92a-3p and wee2 during early embryo development was confirmed by absolute quantification and in situ hybridization. Targeted knockdown of miR-92a-3p in embryos resulted in retarded embryonic development over the first 24 hours. Target validation assays demonstrated that miR-92a-3p interacted with the predicted wee2 3’UTR binding site, which was strongly suppressed by endogenous miR-92a-3p. Our results suggest that miR-92a-3p regulates the abundance of wee2, a cyclin-dependent kinase 1 inhibitor, thus having important role in regulation of the cell cycle during cleavage stages in zebrafish.Summary statementIn zebrafish, maternal miR-92a-3p was demonstrated to suppress translation of wee2, a cyclin-dependent kinase 1 inhibitor which regulates cell cycle progression during the early stages of embryogenesis.

Background The origin and contribution of seminal plasma RNAs into the whole semen RNA repertoire are poorly known, frequently being overlooked or neglected. Virtually nothing is known about seminal plasma RNAs in fish, including small RNAs, which have regulatory functions in gonadal development. Results In this study, we profiled microRNA (miRNA) constituents in the whole semen, as well as in fractionated spermatozoa and seminal plasma of Atlantic salmon (Salmo salar). Among 306 conserved miRNAs, 85 were differentially accumulated (>2 log-fold change and p-value < 0.01) between spermatozoa and the seminal plasma. We identified a number of seminal plasma-enriched and spermatozoa-enriched miRNAs. We localized the expression of some miRNAs in juvenile and mature testes. Two abundant miRNAs, miR-92a-3p and miR-202-5p, localized to both spermatogonia and somatic supporting cells in immature testis, and they were also highly abundant in somatic cells in mature testis. miR-15c-5p, miR-30d-5p, miR-93a-5p, and miR-730-5p were detected only in mature testis. miRs 92a-3p, 202-5p, 15c-5p, and 30d-5p were detected also in a juvenile ovary in locations corresponding to these from the testis. Additional RT-qPCR experiment demonstrated lack of correlation in miRNA transcript levels in seminal plasma versus blood plasma. Conclusions Our results indicate that salmon semen is rich in miRNAs, which are present in both spermatozoa and seminal plasma. The latter ones have partially different profile indicating their heterogenic origin. Testicular supporting somatic cells are likely the source of seminal plasma enrichment, whereas blood plasma unlikely contributes to the seminal plasma miRNA repertoire.

To our knowledge, there is no report on microRNA (miRNA) expression and their target analysis in relation to the type of the first feed and its effect on the further growth of fish. Atlantic cod (Gadus morhua) larvae have better growth and development performance when fed natural zooplankton as a start-feed, as compared with those fed typical aquaculture start-feeds. In our experiment, two groups of Atlantic cod larvae were fed reference feed (zooplankton, mostly copepods, filtered from a seawater pond) v. aquaculture feeds: enriched rotifers (Brachionus sp.) and later brine shrimp (Artemia salina). We examined the miRNA expressions of six defined developmental stages as determined and standardised by body length from first feeding for both diet groups. We found eight miRNA (miR-9, miR-19a, miR-130b, miR-146, miR-181a, miR-192, miR-206 and miR-11240) differentially expressed between the two feeding groups in at least one developmental stage. We verified the next-generation sequencing data using real-time RT-PCR. We found 397 putative targets (mRNA) to the differentially expressed miRNA; eighteen of these mRNA showed differential expression in at least one stage. The patterns of differentially expressed miRNA and their putative target mRNA were mostly inverse, but sometimes also concurrent. The predicted miRNA targets were involved in different pathways, including metabolic, phototransduction and signalling pathways. The results of this study provide new nutrigenomic information on the potential role of miRNA in mediating nutritional effects on growth during the start-feeding period in fish larvae.

Studies in non-teleost vertebrates have found microRNAs (miRNAs) to be essential for proper gonadal development. However, comparatively little is known about their role during gonadal development in teleost fishes. So far in zebrafish, a model teleost, transcript profiling throughout gonadal development has not been established because of a tiny size of an organ in juvenile stages and its poor distinguishability from surrounding tissues. We performed small RNA sequencing on isolated gonads of See-Thru-Gonad line, from the undifferentiated state at 3 weeks post fertilization (wpf) to fully mature adults at 24 wpf. We identified 520 gonadal mature miRNAs; 111 of them had significant changes in abundance over time, while 50 miRNAs were either testis- or ovary-enriched significantly in at least one developmental stage. We characterized patterns of miRNA abundance over time including isomiR variants. We identified putative germline versus gonadal somatic miRNAs through differential small RNA sequencing of isolated gametes versus the whole gonads. This report is the most comprehensive analysis of the miRNA repertoire in zebrafish gonads during the sexual development to date and provides an important database from which functional studies can be performed.

Zebrafish are an important model species in developmental biology. However, their potential in reproductive biology research has yet to be realized. In this study, we establishedSee-Thru-Gonadzebrafish, a transparent line with fluorescently labeled germ cells visible throughout the life cycle, validated its gonadal development features, and demonstrated its applicability by performing a targeted gene knockdown experiment using vivo-morpholinos (VMOs). To establish the line, we crossed the zf45Tg andmitfaw2/w2;mpv17b18/b18zebrafish lines. We documented thein vivovisibility of the germline-specific fluorescent signal throughout development, from gametes through embryonic and juvenile stages up to sexual maturity, and validated gonadal development with histology. We performed targeted gene knockdown of the microRNA (miRNA) miR-92a-3p through injection of VMOs directly to maturing ovaries. After the treatment, zebrafish were bred naturally. Embryos from miR-92a-3p knockdown ovaries had a significant reduction in relative miR-92a-3p expression and a higher percentage of developmental arrest at the 1-cell stage as compared with 5-base mismatch-treated controls. The experiment demonstrates thatSee-Thru-Gonadline can be successfully used for vertical transmission of the effects of targeted gene knockdown in ovaries into their offspring.

Farming of Atlantic cod, Gadus morhua L., has been facing several challenges lately. Biological issues such as differences in egg quality and lack of high quality larvae are considered as prominent limiting factors. The aim of this study was to compare fertilization and hatching rates, incidence of malformed larvae at hatching, larval viability (high-salinity stress and starvation tolerance tests) and the biochemical profile (proteins, amino acids, lipids, fatty acids and minerals) between eggs produced from wild and farmed broodstocks. Twenty-five batches of eggs from farmed broodstock (FB) and 27 egg batches from wild broodstock (WB) were collected during the reproductive season. Fertilization and hatching rates were significantly higher in WB group (91 ± 3% and 86 ± 3%, respectively) than in FB (75 ± 3% and 66 ± 3%, respectively). In high-salinity stress test, WB larvae survived for a significantly longer time than FB larvae. No differences between egg batches from the two broodstocks were observed in starvation tolerance tests, incidence of malformed larvae at hatching, as well as in the total content of proteins, lipids, fatty acids, zinc, and calcium concentrations. Regarding fatty acids, FB eggs had significantly higher levels of C16:0, C18:0, C18:2n − 6, C18:3n − 3, C18:3n − 6, C20:4n − 6, C20n:5n − 3, C22:5n − 3 and C22:5n − 6, whereas WB eggs had significantly higher levels of C14:0, C16:1n − 7, C18:4n − 3, C20:4n − 3, C22:6n − 3 and C24:1n − 9. As for the amino acid profile, WB eggs had significantly higher levels of aspartic acid, serine, glycine, threonine, taurine and leucine, while FB eggs had significantly higher levels of proline, tryptophan, phenylalanine and arginine. The concentrations of minerals such as copper and phosphorus were significantly higher in WB eggs, whereas in FB eggs iron concentration was significantly higher. Overall, the biological parameters indicated that egg and larvae originating from WB were of superior quality than those from FB. The information on the biochemical profile may be useful in improving the quality of broodstock feeds for Atlantic cod.

The role of miRNA in fish sexual development is not elucidated yet. We profiled miRNAs in gonads and brains of Atlantic halibut using SOLiD sequencing technology. We found tissue- and sexually dimorphic expression of several miRNAs, including miR-29a, miR-34, miR-143, miR-145, miR-202-3p, miR-451, and miR-2188. miR-9 and miR-202 were abundant in brain and gonads, respectively. In the next step, we selected some miRNAs showing differential expression patterns between sexes and performed RT-qPCR on 3 age groups: juveniles, 3-year-, and 5-year-olds. In brains, miR-451 was significantly down-regulated in juveniles compared to adults. let-7a, miR-143, and miR-202-3p were up-regulated in gonads of mature males compared to immature females at the same age. We investigated the effect of suppressing aromatase cytochrome P450 enzyme on miRNA expression at the onset of sex differentiation through masculinization with Fadrozole or 17-α-methyltestosterone. We found significant differences in miRNA expression between masculinized individuals and untreated controls. miR-202-3p was significantly down-regulated in female juveniles compared to male juveniles. The expression levels of let-7a and miR-451 were restored after termination of the masculinization treatment. Our data give a first insight into miRNA involvement in sexual development in teleosts.

The Atlantic cod (Gadus morhua) is an emerging aquaculture species. Efforts to develop and characterize its genomic recourses, including draft-grade genome sequencing, have been initiated by the research community. The transcriptome represents the whole complement of RNA transcripts in cells and tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. We are investigating the Atlantic cod transcriptome by Roche 454, Illumina GA, and ABI SOLiD deep sequencing platforms and corresponding bioinformatics. Both embryonic developmental stages and adult tissues are studied. Here we summarize our recent progress in the analyses of nuclear and mitochondrial polyA mRNAs, non-protein-coding intermediate RNAs, and regulatory microRNAs.

Significant efforts have been made to elucidate factors affecting egg quality in fish. Recently, we have shown that eggs originating from wild broodstock (WB) of Atlantic cod (Gadus morhua L.) are of superior quality to those derived from farmed broodstock (FB), and this is associated with differences in the chemical composition of egg yolk. However, maternal transcripts, accumulated during oogenesis, have not been studied extensively in fish. The aim of the present study was to characterize putative maternal mRNA transcriptome in fertilized eggs of Atlantic cod and to compare transcript pools between WB and FB in order to investigate the relation between egg developmental potential and putative maternal mRNA deposits. We performed high-throughput 454 pyrosequencing. For each WB and FB group, five cDNA libraries were individually tagged and sequenced, resulting in 98,687 (WB) and 119,333 (FB) average reads per library. Sequencing reads were de novo assembled, annotated, and mapped. Out of 13,726 identified isotigs, 238 were differentially expressed between WB and FB, with 155 isotigs significantly upregulated in WB. The sequence reads were mapped to 11,340 different Atlantic cod transcripts and 158 sequences were differentially expressed between the 2 groups. Important transcripts involved in fructose metabolism, fatty acid metabolism, glycerophospholipid metabolism, and oxidative phosphorylation were differentially represented between the two broodstock groups, showing potential as biomarkers of egg quality in teleosts. Our findings contribute to the hypothesis that maternal mRNAs affect egg quality and, consequently, the early development of fish.

Background MicroRNAs (miRNAs) play a major role in animal ontogenesis. Size variants of miRNAs, isomiRs, are observed along with the main miRNA types, but their origin and possible biological role are uncovered yet. Developmental profiles of miRNAs have been reported in few fish species only and, to our knowledge, differential expressions of isomiRs have not yet been shown during fish development. Atlantic halibut, Hippoglossus hippoglossus L., undergoes dramatic metamorphosis during early development from symmetrical pelagic larval stage to unsymmetrical flatfish. No data exist on role of miRNAs in halibut metamorphosis. Results miRNA profiling using SOLiD deep sequencing technology revealed a total of 199 conserved, one novel antisense, and one miRNA* mature form. Digital expression profiles of selected miRNAs were validated using reverse transcription quantitative PCR. We found developmental transition-specific miRNA expression. Expression of some miRNA* exceeded the guide strand miRNA. We revealed that nucleotide truncations and/or additions at the 3' end of mature miRNAs resulted in size variants showing differential expression patterns during the development in a number of miRNA families. We confirmed the presence of isomiRs by cloning and Sanger sequencing. Also, we found inverse relationship between expression levels of sense/antisense miRNAs during halibut development. Conclusion Developmental transitions during early development of Atlantic halibut are associated with expression of certain miRNA types. IsomiRs are abundant and often show differential expression during the development.

SUMMARY In Senegalese sole (Solea senegalensis Kaup), growth is negatively correlated to dietary lipid levels. To understand the molecular basis of this effect a molecular toolbox of 12 genes, including fgf6, fst, mstn1, myf5, mrf4, myod1, myod2, myog, myHC, mylc2, igf1r and insr, was developed. The expression profiles of these genes were investigated in white muscle and liver of fish fed with three dietary lipid levels (4%, 12% and 20%). The expression of igf-I and igf-II was also examined. MRFs and myosins were only expressed in the muscle and, except for myf5, the general trend was a decrease in expression with an increase in dietary lipids. Fgf6 was identified for the first time in liver and its expression augmented in hepatic tissues with increasing dietary lipid levels. A similar tendency was observed for mstn1 and igf-I. The opposite was observed for igf1r expression in muscle and liver. Myog, mrf4, mylc2 and igf1r were highly correlated with growth and nutrient utilisation indices. In addition to its practical implications, this work provides a valuable contribution towards our understanding of the genetic networks controlling growth in teleosts.