Ocean Acidification Disrupts Prey Responses to Predator Cues but Not Net Prey Shell Growth in Concholepas concholepas (loco)

Background Most research on Ocean Acidification (OA) has largely focused on the process of calcification and the physiological trade-offs employed by calcifying organisms to support the building of calcium carbonate structures. However, there is growing evidence that OA can also impact upon other key biological processes such as survival, growth and behaviour. On wave-swept rocky shores the ability of gastropods to self-right after dislodgement, and rapidly return to normal orientation, reduces the risk of predation. Methodology/Principal Findings The impacts of OA on this self-righting behaviour and other important parameters such as growth, survival, shell dissolution and shell deposition in Concholepas concholepas (loco) were investigated under contrasting pCO2 levels. Although no impacts of OA on either growth or net shell calcification were found, the results did show that OA can significantly affect self-righting behaviour during the early ontogeny of this species with significantly faster righting times recorded for individuals of C. concholepas reared under increased average pCO2 concentrations (± SE) (716±12 and 1036±14 µatm CO2) compared to those reared at concentrations equivalent to those presently found in the surface ocean (388±8 µatm CO2). When loco were also exposed to the predatory crab Acanthocyclus hassleri, righting times were again increased by exposure to elevated CO2, although self-righting times were generally twice as fast as those observed in the absence of the crab. Conclusions and Significance These results suggest that self-righting in the early ontogeny of C. concholepas will be positively affected by pCO2 levels expected by the end of the 21st century and beginning of the next one. However, as the rate of self-righting is an adaptive trait evolved to reduce lethal predatory attacks, our result also suggest that OA may disrupt prey responses to predators in nature

Circulating microRNAs Reveal Time Course of Organ Injury in a Porcine Model of Acetaminophen-Induced Acute Liver Failure

Acute liver failure is a rare but catastrophic condition which can progress rapidly to multi-organ failure. Studies investigating the onset of individual organ injury such as the liver, kidneys and brain during the evolution of acute liver failure, are lacking. MicroRNAs are short, non-coding strands of RNA that are released into the circulation following tissue injury. In this study, we have characterised the release of both global microRNA and specific microRNA species into the plasma using a porcine model of acetaminophen-induced acute liver failure. Pigs were induced to acute liver failure with oral acetaminophen over 19h±2h and death occurred 13h±3h thereafter. Global microRNA concentrations increased 4h prior to acute liver failure in plasma (P<0.0001) but not in isolated exosomes, and were associated with increasing plasma levels of the damage-associated molecular pattern molecule, genomic DNA (P<0.0001). MiR122 increased around the time of onset of acute liver failure (P<0.0001) and was associated with increasing international normalised ratio (P<0.0001). MiR192 increased 8h after acute liver failure (P<0.0001) and was associated with increasing creatinine (P<0.0001). The increase in miR124-1 occurred concurrent with the pre-terminal increase in intracranial pressure (P<0.0001) and was associated with decreasing cerebral perfusion pressure (P<0.002)

miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.</p> <p>Results</p> <p>Here, we present the miR-Q assay as a highly sensitive quantitative reverse transcription PCR (qRT-PCR) for expression analysis of small RNAs such as miRNA molecules. It shows a high dynamic range of 6 to 8 orders of magnitude comprising a sensitivity of up to 0.2 fM miRNA, which corresponds to single copies per cell. There is nearly no cross reaction among closely-related miRNA family members, which points to the high specificity of the assays. Using this approach, we quantified the expression of let-7b in different human cell lines as well as miR-145 and miR-21 expression in porcine intestinal samples.</p> <p>Conclusion</p> <p>miR-Q is a cost-effective and highly specific approach, which neither requires the use of fluorochromic probes, nor Locked Nucleic Acid (LNA)-modified oligonucleotides. Moreover, it provides a remarkable increase in specificity and simplified detection of small RNAs.</p

Plasma miRNA as Biomarkers for Assessment of Total-Body Radiation Exposure Dosimetry

The risk of radiation exposure, due to accidental or malicious release of ionizing radiation, is a major public health concern. Biomarkers that can rapidly identify severely-irradiated individuals requiring prompt medical treatment in mass-casualty incidents are urgently needed. Stable blood or plasma-based biomarkers are attractive because of the ease for sample collection. We tested the hypothesis that plasma miRNA expression profiles can accurately reflect prior radiation exposure. We demonstrated using a murine model that plasma miRNA expression signatures could distinguish mice that received total body irradiation doses of 0.5 Gy, 2 Gy, and 10 Gy (at 6 h or 24 h post radiation) with accuracy, sensitivity, and specificity of above 90%. Taken together, these data demonstrate that plasma miRNA profiles can be highly predictive of different levels of radiation exposure. Thus, plasma-based biomarkers can be used to assess radiation exposure after mass-casualty incidents, and it may provide a valuable tool in developing and implementing effective countermeasures

Functionally Orthologous Viral and Cellular MicroRNAs Studied by a Novel Dual-Fluorescent Reporter System

Recent research raised the possibility that some viral microRNAs (miRNAs) may function as orthologs of cellular miRNAs. In the present work, to study the functional orthologous relationships of viral and cellular miRNAs, we first constructed a dual-fluorescent protein reporter vector system for the easy determination of miRNA function. By expressing the miRNAs and the indicator and internal control fluorescent proteins individually from a single vector, this simple reporter system can be used for miRNA functional assays that include visualizing miRNA activity in live cells. Sequence alignments indicated that the simian virus 40 (SV40) encoded miRNA sv40-mir-S1-5p contains a seed region identical to that of the human miRNA hsa-miR423-5p. Using the new reporter system, it was found that sv40-mir-S1-5p and hsa-miR423-5p downregulate the expression of common artificial target mRNAs and some predicted biological targets of hsa-miR423-5p, demonstrating that they are functional orthologs. The human immunodeficiency virus 1 (HIV-1) encoded hiv1-miR-N367 also contains a seed sequence identical to that of the human miRNA hsa-miR192. Functional assays showed that hiv1-miR-N367 and hsa-miR192 could downregulate common artificial and predicted biological targets, suggesting that these miRNAs may also act as functional orthologs. Thus, this study presents a simple and universal system for testing miRNA function and identifies two new pairs of functional orthologs, sv40-mir-S1-5p and hsa-miR423-5p as well as hiv-1-miR-N367 and hsa-miR192. These findings also expand upon our current knowledge of functional homology and imply that a more general phenomenon of orthologous relationships exists between viral and cellular miRNAs

Molecular signatures of maturing dendritic cells: implications for testing the quality of dendritic cell therapies

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DCs) are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) induction in order to characterize the usefulness of mature DCs (mDCs) for immune therapy and to identify biomarkers for assessing the quality of mDCs.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-γ and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis.</p> <p>Results</p> <p>After 24 hours of LPS and IFN-γ stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs.</p> <p>Conclusion</p> <p>DCs, matured with LPS and IFN-γ, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.</p

Suppression of microRNA-31 increases sensitivity to 5-FU at an early stage, and affects cell migration and invasion in HCT-116 colon cancer cells

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are endogenously expressed noncoding RNAs with important biological and pathological functions. Although several studies have shown that microRNA-31 (miR-31) is obviously up-regulated in colorectal cancer (CRC), there is no study on the functional roles of miR-31 in CRC.</p> <p>Methods</p> <p>Anti-miR™ miRNA 31 inhibitor (anti-miR-31) is a sequence-specific and chemically modified oligonucleotide to specifically target and knockdown miR-31 molecule. The effect of anti-miR-31 transfection was investigated by real-time PCR. HCT-116^p53+/+and HCT-116^p53-/-colon cancer cells were treated by anti-miR-31 with or without 5-fluorouracil (5-FU), cell proliferation was determined by MTT assay; apoptosis was detected by DAPI staining; cell cycle was evaluated by flow cytometry; colony formation, migration and invasion assays were performed to investigate the effect of suppression of miR-31 on the cell lines.</p> <p>Results</p> <p>Real-time PCR results showed that anti-miR-31 was efficiently introduced into the cells and reduced miR-31 levels to 44.1% in HCT-116^p53+/+and 67.8% in HCT-116^p53-/-cell line (<it>p </it>= 0.042 and 0.046). MTT results showed that anti-miR-31 alone had no effect on the proliferation of HCT-116^p53+/+or HCT-116^p53-/-. However, when combined with 5-FU, anti-miR-31 inhibited the proliferation of the two cell lines as early as 24 h after exposure to 5-FU (<it>p </it>= 0.038 and 0.044). Suppression of miR-31 caused a reduction of the migratory cells by nearly 50% compared with the negative control in both HCT-116^p53+/+and HCT-116^p53-/-(<it>p </it>= 0.040 and 0.001). The invasive ability of the cells were increased by 8-fold in HCT-116^p53+/+and 2-fold in HCT-116^p53-/-(<it>p </it>= 0.045 and 0.009). Suppression of miR-31 had no effect on cell cycle and colony formation (<it>p </it>> 0.05).</p> <p>Conclusions</p> <p>Suppression of miR-31 increases sensitivity to 5-FU at an early stage, and affects cell migration and invasion in HCT-116 colon cancer cells.</p

Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): Their expression during early embryonic development

<p>Abstract</p> <p>Background</p> <p>Current literature and our previous results on expression patterns of oocyte-specific genes and transcription factors suggest a global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). MicroRNAs (miRNAs) are small, non-coding regulatory RNAs (19–23 nucleotides) that regulate gene expression by guiding target mRNA cleavage or translational inhibition. These regulatory RNAs are potentially involved in the degradation of maternally inherited mRNAs during early embryogenesis.</p> <p>Results</p> <p>To identify miRNAs that might be important for early embryogenesis in rainbow trout, we constructed a miRNA library from a pool of unfertilized eggs and early stage embryos. Sequence analysis of random clones from the library identified 14 miRNAs, 4 of which are novel to rainbow trout. Real-time PCR was used to measure the expression of all cloned miRNAs during embryonic development. Four distinct expression patterns were observed and some miRNAs showed up-regulated expression during EGA. Analysis of tissue distribution of these miRNAs showed that some are present ubiquitously, while others are differentially expressed among different tissues. We also analyzed the expression patterns of Dicer, the enzyme required for the processing of miRNAs and Stat3, a transcription factor involved in activating the transcription of miR-21. Dicer is abundantly expressed during EGA and Stat3 is up-regulated before the onset of EGA.</p> <p>Conclusion</p> <p>This study led to the discovery of 14 rainbow trout miRNAs. Our data support the notion that Dicer processes miRNAs and Stat3 induces expression of miR-21 and possibly other miRNAs during EGA. These miRNAs in turn guide maternal mRNAs for degradation, which is required for normal embryonic development.</p

Interchromosomal Duplications on the Bactrocera oleae Y Chromosome Imply a Distinct Evolutionary Origin of the Sex Chromosomes Compared to Drosophila

BACKGROUND: Diptera have an extraordinary variety of sex determination mechanisms, and Drosophila melanogaster is the paradigm for this group. However, the Drosophila sex determination pathway is only partially conserved and the family Tephritidae affords an interesting example. The tephritid Y chromosome is postulated to be necessary to determine male development. Characterization of Y sequences, apart from elucidating the nature of the male determining factor, is also important to understand the evolutionary history of sex chromosomes within the Tephritidae. We studied the Y sequences from the olive fly, Bactrocera oleae. Its Y chromosome is minute and highly heterochromatic, and displays high heteromorphism with the X chromosome. METHODOLOGY/PRINCIPAL FINDINGS: A combined Representational Difference Analysis (RDA) and fluorescence in-situ hybridization (FISH) approach was used to investigate the Y chromosome to derive information on its sequence content. The Y chromosome is strewn with repetitive DNA sequences, the majority of which are also interdispersed in the pericentromeric regions of the autosomes. The Y chromosome appears to have accumulated small and large repetitive interchromosomal duplications. The large interchromosomal duplications harbour an importin-4-like gene fragment. Apart from these importin-4-like sequences, the other Y repetitive sequences are not shared with the X chromosome, suggesting molecular differentiation of these two chromosomes. Moreover, as the identified Y sequences were not detected on the Y chromosomes of closely related tephritids, we can infer divergence in the repetitive nature of their sequence contents. CONCLUSIONS/SIGNIFICANCE: The identification of Y-linked sequences may tell us much about the repetitive nature, the origin and the evolution of Y chromosomes. We hypothesize how these repetitive sequences accumulated and were maintained on the Y chromosome during its evolutionary history. Our data reinforce the idea that the sex chromosomes of the Tephritidae may have distinct evolutionary origins with respect to those of the Drosophilidae and other Dipteran families

The Smallest Known Genomes of Multicellular and Toxic Cyanobacteria: Comparison, Minimal Gene Sets for Linked Traits and the Evolutionary Implications

Cyanobacterial morphology is diverse, ranging from unicellular spheres or rods to multicellular structures such as colonies and filaments. Multicellular species represent an evolutionary strategy to differentiate and compartmentalize certain metabolic functions for reproduction and nitrogen (N2) fixation into specialized cell types (e.g. akinetes, heterocysts and diazocytes). Only a few filamentous, differentiated cyanobacterial species, with genome sizes over 5 Mb, have been sequenced. We sequenced the genomes of two strains of closely related filamentous cyanobacterial species to yield further insights into the molecular basis of the traits of N2 fixation, filament formation and cell differentiation. Cylindrospermopsis raciborskii CS-505 is a cylindrospermopsin-producing strain from Australia, whereas Raphidiopsis brookii D9 from Brazil synthesizes neurotoxins associated with paralytic shellfish poisoning (PSP). Despite their different morphology, toxin composition and disjunct geographical distribution, these strains form a monophyletic group. With genome sizes of approximately 3.9 (CS-505) and 3.2 (D9) Mb, these are the smallest genomes described for free-living filamentous cyanobacteria. We observed remarkable gene order conservation (synteny) between these genomes despite the difference in repetitive element content, which accounts for most of the genome size difference between them. We show here that the strains share a specific set of 2539 genes with >90% average nucleotide identity. The fact that the CS-505 and D9 genomes are small and streamlined compared to those of other filamentous cyanobacterial species and the lack of the ability for heterocyst formation in strain D9 allowed us to define a core set of genes responsible for each trait in filamentous species. We presume that in strain D9 the ability to form proper heterocysts was secondarily lost together with N2 fixation capacity. Further comparisons to all available cyanobacterial genomes covering almost the entire evolutionary branch revealed a common minimal gene set for each of these cyanobacterial traits