Potential Biomarkers for the Earlier Diagnosis of Kidney and Liver Damage in Acute Intermittent Porphyria.

Acute intermittent porphyria (AIP) is an inherited metabolic disorder associated with complications including kidney failure and hepatocellular carcinoma, probably caused by elevations in the porphyrin precursors porphobilinogen (PBG) and delta-aminolevulinic acid (ALA). This study explored differences in modern biomarkers for renal and hepatic damage between AIP patients and controls. Urine PBG testing, kidney injury panels, and liver injury panels, including both routine and modern biomarkers, were performed on plasma and urine samples from AIP cases and matched controls (50 and 48 matched pairs, respectively). Regarding the participants' plasma, the AIP cases had elevated kidney injury marker-1 (KIM-1, p = 0.0002), fatty acid-binding protein-1 (FABP-1, p = 0.04), and α-glutathione S-transferase (α-GST, p = 0.001) compared to the matched controls. The AIP cases with high PBG had increased FABP-1 levels in their plasma and urine compared to those with low PBG. In the AIP cases, KIM-1 correlated positively with PBG, CXCL10, CCL2, and TCC, and the liver marker α-GST correlated positively with IL-13, CCL2, and CCL4 (all p < 0.05). In conclusion, KIM-1, FABP-1, and α-GST could represent potential early indicators of renal and hepatic damage in AIP, demonstrating associations with porphyrin precursors and inflammatory markers.

Dental and Periodontal Health in Acute Intermittent Porphyria.

In the inherited metabolic disorder acute intermittent porphyria (AIP), high sugar intake prevents porphyric attacks due to the glucose effect and the following high insulin levels that may lower AIP disease activity. Insulin resistance is a known risk factor for periodontitis and sugar changes diabetogenic hormones and affects dental health. We hypothesized differences in homeostasis model assessment (HOMA) scores for insulin resistance in AIP cases vs. controls and in those with periodontitis. Our aim was to systematically study dental health in AIP as poor dental health was previously only described in case reports. Further, we aimed to examine if poor dental health and kidney failure might worsen AIP as chronic inflammation and kidney failure might increase disease activity. In 47 AIP cases and 47 matched controls, X-rays and physical examination of clinical attachment loss (CAL), probing pocket depth (PPD), and decayed missing filled teeth (DMFT) were performed. Dietary intake was evaluated through a diet logbook. Plasma cytokines and diabetogenic hormones were measured using multiplex technology and urine porphobilinogen and kidney and liver function by routine methods. An excel spreadsheet from the University of Oxford was used to estimate HOMA scores; beta cell function, HOMA%B (%B), insulin sensitivity, HOMA%S (%S), and insulin resistance HOMA-IR (IR), based on glucose and plasma (P) C-peptide. The Wilcoxon matched-pairs signed rank test, the Mann−Whitney U-test, and Spearman's non-parametric correlation were used. Insulin (p = 0.007) and C-peptide (p = 0.006) were higher in the AIP cases with periodontitis versus those without. In AIP patients, the liver fibrosis index 4 correlated with DMFT (p < 0.001) and CAL ≥4 mm (p = 0.006); the estimated glomerular filtration rate correlated with DMFT (p < 0.001) and CAL ≥4 mm (p = 0.02). CAL ≥4 mm was correlated with chemokine ligand 11 and interleukin (IL)-13 (p = 0.04 for both), and PPD >5 mm was correlated with plasminogen activator inhibitor-1 (p = 0.003) and complement component 3 (p = 0.02). In conclusion, dental health in AIP cases was correlated with insulin resistance, inflammatory markers, and biomarkers of kidney and liver function, demonstrating that organ damage in the kidney and liver are associated with poorer dental health.

Blood Transcriptome Analysis of Septic Patients Reveals a Long Non-Coding Alu-RNA in the Complement C5a Receptor 1 Gene.

Many severe inflammation conditions are complement-dependent with the complement component C5a-C5aR1 axis as an important driver. At the RNA level, the blood transcriptome undergoes programmed expression of coding and long non-coding RNAs to combat invading microorganisms. Understanding the expression of long non-coding RNAs containing Alu elements in inflammation is important for reconstructing cell fate trajectories leading to severe disease. We have assembled a pipeline for computation mining of new Alu-containing long non-coding RNAs by intersecting immune genes with known Alu coordinates in the human genome. By applying the pipeline to patient bulk RNA-seq data with sepsis, we found immune genes containing 48 Alu insertion as robust candidates for further study. Interestingly, 1 of the 48 candidates was located within the complement system receptor gene C5aR1 and holds promise as a target for RNA therapeutics.

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood.

Introduction: Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy. Methods: In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy. Results and Discussion: We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.

A mitochondrial long noncoding RNA in atlantic cod harbors complex heteroplasmic tandem repeat motifs.

A heteroplasmic tandem repeat (HTR) array occupies 100 to 300 bp of the mitochondrial DNA control region in the Atlantic cod, and recently we noted that the repeat appeared integrated in a polyadenylated mitochondrial long noncoding RNA. Here we provide a more detailed analysis of the mitochondrial HTR in the mitochondrial genome of 134 Atlantic cod specimens. We report all specimens to harbor mitochondrial HTRs in the control region, and identified 26 distinct variants among the 402 repeat motifs assessed. Whereas most specimens contained HTR profiles of 2-5 copies consisting of the same 40-bp motif, 22 specimens showed compound HTR arrays of at least two types of motifs present in the same mitochondrial DNA molecule. We found HTR profiles to be highly conserved between different tissue types of a single individual, and strictly maternally inherited in a mating experiment between parental Atlantic cod expressing different HTR profiles and array motifs. We conclude that mitochondrial heteroplasmy in the control region is very common in Atlantic cod, and results in length heterogenity of the long noncoding RNA lncCR-H.

Mitochondrial genome variation of Atlantic cod.

OBJECTIVE: The objective of this study was to analyse intraspecific sequence variation of Atlantic cod mitochondrial DNA, based on a comprehensive collection of completely sequenced mitochondrial genomes. RESULTS: We determined the complete mitochondrial DNA sequence of 124 cod specimens from the eastern and western part of the species' distribution range in the North Atlantic Ocean. All specimens harboured a unique mitochondrial DNA haplotype. Nine hundred and fifty-two polymorphic sites were identified, including 109 non-synonymous sites within protein coding regions. Eighteen variable sites were identified as indels, exclusively distributed in structural RNA genes and non-coding regions. Phylogeographic analyses based on 156 available cod mitochondrial genomes did not reveal a clear structure. There was a lack of mitochondrial genetic differentiation between two ecotypes of cod in the eastern North Atlantic, but eastern and western cod were differentiated and mitochondrial genome diversity was higher in the eastern than the western Atlantic, suggesting deviating population histories. The geographic distribution of mitochondrial genome variation seems to be governed by demographic processes and gene flow among ecotypes that are otherwise characterized by localized genomic divergence associated with chromosomal inversions.

Elucidating the Small Regulatory RNA Repertoire of the Sea Anemone Anemonia viridis Based on Whole Genome and Small RNA Sequencing.

Cnidarians harbor a variety of small regulatory RNAs that include microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), but detailed information is limited. Here, we report the identification and expression of novel miRNAs and putative piRNAs, as well as their genomic loci, in the symbiotic sea anemone Anemonia viridis. We generated a draft assembly of the A. viridis genome with putative size of 313 Mb that appeared to be composed of about 36% repeats, including known transposable elements. We detected approximately equal fractions of DNA transposons and retrotransposons. Deep sequencing of small RNA libraries constructed from A. viridis adults sampled at a natural CO2 gradient off Vulcano Island, Italy, identified 70 distinct miRNAs. Eight were homologous to previously reported miRNAs in cnidarians, whereas 62 appeared novel. Nine miRNAs were recognized as differentially expressed along the natural seawater pH gradient. We found a highly abundant and diverse population of piRNAs, with a substantial fraction showing ping-pong signatures. We identified nearly 22% putative piRNAs potentially targeting transposable elements within the A. viridis genome. The A. viridis genome appeared similar in size to that of other hexacorals with a very high divergence of transposable elements resembling that of the sea anemone genus Exaiptasia. The genome encodes and expresses a high number of small regulatory RNAs, which include novel miRNAs and piRNAs. Differentially expressed small RNAs along the seawater pH gradient indicated regulatory gene responses to environmental stressors.

Sea anemones possess dynamic mitogenome structures.

A notable feature of hexacoral mitogenomes is the presence of complex self-catalytic group I introns. We investigated mitogenome structural variations and evolutionary mechanisms in actiniarian sea anemones based on the complete mitogenome sequence of the cold-water sea anemone species Urticina eques, Bolocera tuediae, Hormathia digitata and Metridium senile, and two isolates of the sub-tropical Aiptasia pulchella. Whole genome sequencing at 50 times coverage of B. tuediae and H. digitata indicated low mtDNA copy number of per haploid nuclear genome and presence of rare haplotypes. A group I intron inserted in ND5 was found to host essential mitochondrial protein genes in all species, and an additional truncated copy of ND5 in B. tuediae. A second group I intron (inserted in COI) that contained a homing endonuclease gene (HEG) was present in all mtDNA examined. Different variants of HEGs were observed, and included expressed elements fused in-frame with upstream exons and free-standing HEGs embedded within the intron. A notable hallmark of HEGs was a high extent of overlap with ribozyme structural elements; the U. eques HEG overlapped with the entire intron. We reconstructed the evolutionary history of the COI intron from insertion at unoccupied cognate sites, through HEG degradation, to intron loss. We also identified a novel insertion element in U. eques that contained two expressed protein-coding genes. An evolutionary analysis of the sea anemone mtDNA genes revealed higher substitution rates in the HEG and the insertion sequence as compared to the other loci, indicating relaxed selective pressures in these elements. We conclude that sea anemone mitogenomes are surprisingly dynamic in structure despite the economical organization and low sequence mutation rate.

A novel beta-defensin antimicrobial peptide in Atlantic cod with stimulatory effect on phagocytic activity.

A novel defensin antimicrobial peptide gene was identified in Atlantic cod, Gadus morhua. This three exon/two intron defensin gene codes for a peptide precursor consisting of two domains: a signal peptide of 26 amino acids and a mature peptide of 40 residues. The mature cod defensin has six conserved cysteine residues that form 1-5, 2-4 and 3-6 disulphide bridges. This pattern is typical of beta-defensins and this gene was therefore named cod beta-defensin (defb). The tertiary structure of Defb exhibits an α/ß fold with one α helix and ß1ß2ß3 sheets. RT-PCR analysis indicated that defb transcripts were present mainly in the swim bladder and peritoneum wall but could also be detected at moderate to low levels in skin, head- and excretory kidneys. In situ hybridisation revealed that defb was specifically expressed by cells located in the swim bladder submucosa and the oocytes. During embryonic development, defb gene transcripts were detectable from the golden eye stage onwards and their expression was restricted to the swim bladder and retina. Defb was differentially expressed in several tissues following antigenic challenge with Vibrio anguillarum, being up-regulated up to 25-fold in head kidney. Recombinant Defb displayed antibacterial activity, with a minimal inhibitory concentration of 0.4-0.8 µM and 25-50 µM against the Gram-(+) bacteria Planococcus citreus and Micrococcus luteus, respectively. In addition, Defb stimulated phagocytic activity of cod head kidney leucocytes in vitro. These findings imply that beta-defensins may play an important role in the innate immune response of Atlantic cod.

Digital marine bioprospecting: mining new neurotoxin drug candidates from the transcriptomes of cold-water sea anemones.

Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries.

Mitogenome polymorphism in a single branch sample revealed by SOLiD deep sequencing of the Lophelia pertusa coral genome.

We present an initial genomic analysis of the non-symbiotic scleractinian coral Lophelia pertusa, the dominant cold-water reef-building coral species in the North Atlantic Ocean. A significant fraction of the deep sequencing reads was of mitochondrial and microbial origins. SOLiD deep sequencing reads from fragment library experiments of total DNA and PCR amplified mitogenome generated about 21,000 times and 136,000 times coverage, respectively, of the 16,150 bp mitogenome. Five polymorphic sites that include two non-synonymous sites in the NADH dehydrogenase subunit 5 genes were detected in both experiments. This observation is surprising since anthozoans in general exhibit very low mtDNA sequence variation at intraspecific level compared to nuclear sequences. More than fifty bacterial species associated with the coral isolate were also sequence detected, representing at least ten complete genomes. Most reads, however, were predicted to originate from the Lophelia nuclear genome.

Expansion of cyclin D and CDK1 paralogs in Oikopleura dioica, a chordate employing diverse cell cycle variants.

Proliferative and endoreduplicative cell cycles are used to variable extents during the ontogeny of individual organisms and in different evolutionary lineages. Chordate growth and development is typically dominated by proliferative cycles, but the urochordate, Oikopleura dioica, has systemically elaborated a number of endocycling modes to support rapid development and growth in an extraordinarily short chordate life cycle. Here, we identify the O. dioica cyclin and cyclin-dependent kinase (CDK) complements and assess their deployment with respect to mitotic, meiotic, and endoreduplicative life cycle phases. Oikopleura dioica's "transcriptional" cyclin and CDK complements are similar to other complex invertebrates, whereas both the "cell cycle" cyclin and CDK complements display astonishing amplifications centered on the cyclin D, cyclin B, and CDK1 families. Somatic endocycles in O. dioica involve downregulation of cyclins B and A, as in other endocycle model systems, but are also characterized by overlapping expression of an array of cyclin D isoforms. Amplification of the mitotic CDK1 family to five paralogs, which continue to be expressed in endocycling phases, is unexpected as suppression of CDK1 activity is central to endocycle transitions in Drosophila and mammals. This amplification is unique among metazoans, and substitutions in odCDK1 paralogs in the nearly invariant cyclin interaction PSTAIRE helix show striking parallels to those in the only other known eukaryotic CDK1 paralogs, plant CDKA and CDKB. As plant CDK1 paralogs exhibit an expanded repertoire of cyclin partners, including cyclin D, the evolutionary coexpansion of odCDK1 and odCyclin D families suggests that multiple CDK1-cyclin D complexes may modulate spatiotemporal control of kinase activity and substrate specificity in diverse cell cycle variants.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron.

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3' end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.

Characterization of mitochondrial mRNAs in codfish reveals unique features compared to mammals.

Expression and processing of mitochondrial gene transcripts are fundamental to mitochondrial function, but information from early vertebrates like teleost fishes is essentially lacking. We have analyzed mitogenome sequences of ten codfishes (family Gadidae), and provide complete sequences from three new species (Saithe, Pollack and Blue whiting). Characterization of the mitochondrial mRNAs in Saithe and Atlantic cod identified a set of ten poly(A) transcripts, and six UAA stop codons are generated by posttranscriptional polyadenylation. Structural assessment of poly(A) sites is consistent with an RNaseP cleavage activity 5' of tRNA acceptor-like stems. COI, ND5 and ND6 mRNAs were found to harbor 3' UTRs with antisense potential extending into neighboring gene regions. While the 3' UTR of COI mRNA is complementary to the tRNA(Ser UCN) and highly similar to that detected in human mitochondria, the ND5 and ND6 3' UTRs appear more heterogenic. Deep sequencing confirms expression of all mitochondrial mRNAs and rRNAs, and provides information about the precise 5' ends in mature transcripts. Our study supports an overall evolutionary conservation in mitochondrial RNA processing events among vertebrates, but reveals some unique 5' and 3' end characteristics in codfish mRNAs with implications to antisense regulation of gene expression.

RNA deep sequencing of the Atlantic cod transcriptome.

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.

Approaching marine bioprospecting in hexacorals by RNA deep sequencing.

RNA deep sequencing represents a new complementary approach in marine bioprospecting. Next-generation sequencing platforms have recently been developed for de novo whole transcriptome analysis, small RNA discovery and gene expression profiling. Deep sequencing transcriptomics (sequencing the complete set of cellular transcripts at a specific stage or condition) leads to sequential identification of all expressed genes in a sample. When combined to high-throughput bioinformatics and protein synthesis, RNA deep sequencing represents a new powerful approach in gene product discovery and bioprospecting. Here we summarize recent progress in the analyses of hexacoral transcriptomes with the focus on cold-water sea anemones and related organisms.

Large-scale sequence analyses of Atlantic cod.

The Atlantic cod (Gadus morhua) is a key species in the North Atlantic ecosystem and commercial fisheries, with increasing aquacultural production in several countries. A Norwegian effort to sequence the complete 0.9Gbp genome by the 454 pyrosequencing technology has been initiated and is in progress. Here we review recent progress in large-scale sequence analyses of the nuclear genome, the mitochondrial genome and genome-wide microRNA identification in the Atlantic cod. The nuclear genome will be de novo sequenced with 25 times oversampling. A total of 120 mitochondrial genomes, sampled from several locations in the North Atlantic, are being completely sequenced by Sanger technology in a high-throughput pipeline. These sequences will be included in a new database for maternal marker reference of Atlantic cod diversity. High-throughput 454 sequencing, as well as Evolutionary Image Array (EvoArray) informatics, is used to investigate the complete set of expressed microRNAs and corresponding mRNA targets in various developmental stages and tissues. Information about microRNA profiles will be essential in the understanding of transcriptome complexity and regulation. Finally, developments and perspectives of Atlantic cod aquaculture are discussed in the light of next-generation high-throughput sequence technologies.

PML-nuclear bodies accumulate DNA in response to polyomavirus BK and simian virus 40 replication.

Promyelocytic nuclear bodies (PML-NBs) are distinct nuclear structures that are involved in apoptosis, differentiation, transcriptional regulation and DNA damage response. These bodies have also been shown to associate with nuclear sites of viral DNA replication. In the present study, we used BrdU pulse labeling to demonstrate that PML-NBs accumulate newly synthesized DNA in cells infected by the polyomaviruses simian virus 40 (SV40) or polyomavirus BK (BKV). Sequestration of DNA molecules in these structures depended on active viral DNA replication, and was observed exclusively in cells that contained prominent viral replication domains. Furthermore, a significant portion of the accumulated DNA was found to be single-stranded, indicating that the sequestered DNA had been subjected to processing by nuclease or DNA unwinding activities. siRNA-mediated suppression of the PML protein prevented the recruitment of single-stranded DNA into nuclear foci, but did not significantly affect the overall efficiency of viral DNA replication. These results indicate a role of PML and PML-NBs in post-replication DNA processing, and suggest that PML-NBs become linked to sites of viral DNA synthesis due to a role of these structures in DNA metabolism.