Symmetric expression of ohnologs encoding conserved antiviral responses in tetraploid common carp suggest absence of subgenome dominance after whole genome duplication.

Allopolyploids often experience subgenome dominance, with one subgenome showing higher levels of gene expression and greater gene retention. Here, we address the functionality of both subgenomes of allotetraploid common carp (Cyprinus carpio) by analysing a functional network of interferon-stimulated genes (ISGs) crucial in anti-viral immune defence. As an indicator of subgenome dominance we investigated retainment of a core set of ohnologous ISGs. To facilitate our functional genomic analysis a high quality genome was assembled (WagV4.0). Transcriptome data from an in vitro experiment mimicking a viral infection was used to infer ISG expression. Transcriptome analysis confirmed induction of 88 ISG ohnologs on both subgenomes. In both control and infected states, average expression of ISG ohnologs was comparable between the two subgenomes. Also, the highest expressing and most inducible gene copies of an ohnolog pair could be derived from either subgenome. We found no strong evidence of subgenome dominance for common carp.

Differential gene expression in narrow- and broad-headed European glass eels (Anguilla anguilla) points to a transcriptomic link of head shape dimorphism with growth rate and chemotaxis.

One of the major challenges in evolutionary biology is to understand the mechanisms underlying morphological dimorphism and plasticity, including the genomic basis of traits and links to ecology. At the yellow eel stage of the European eel (Anguilla anguilla), two morphotypes are found: broad- and narrow-heads. This dimorphism has been linked to dietary differences, with broad-heads feeding on harder, larger prey than narrow-heads. However, recent research showed that both morphotypes could be distinguished at the glass eel stage, the nonfeeding predecessor of the yellow eel stage, implying that nondietary factors play a role in the development of this head shape dimorphism. Here, we used transcriptome profiling (RNAseq) to identify differentially expressed genes between broad- and narrow-headed glass eels. We found 260 significantly differentially expressed genes between the morphotypes, of which most were related to defence and immune responses. Interestingly, two genes involved in growth (soma and igf2) were significantly upregulated in narrow-heads, while nine genes involved in chemotaxis showed significant differential expression. Thus, we found support for the observation that head shape is associated with somatic growth, with fast-growing eels developing a narrower head. Additionally, observations in the wild have shown that slow-growers prefer freshwater, while fast-growers prefer brackish water. The differential expression of genes involved in chemotaxis seems to indicate that glass eel growth rate and habitat choice are linked. We hypothesize that two levels of segregation could take place in the European eel: first according to habitat choice and second according to feeding preference.

A full-body transcriptome and proteome resource for the European common carp.

BACKGROUND: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. RESULTS: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. CONCLUSIONS: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

In- and outdoor reproduction of first generation common sole Solea solea under a natural photothermal regime: Temporal progression of sexual maturation assessed by monitoring plasma steroids and gonadotropin mRNA expression.

Reproduction of many temperate fishes is seasonal and maturation and spawning of gametes are under photothermal control. Reproductive success of first generation (G1) common sole Solea solea in captivity has been low. In this study, the sexual maturation status has been assessed during the prespawning months in G1 sole that were housed (a) outdoor under the natural photoperiod and temperature, or (b) indoor under artificial photothermal induction. Maturation was assessed in male and female G1 broodstock in November as controls, after which the remaining population was divided over two outdoor flow-through tanks placed in a pond and two indoor recirculating aquaculture system (RAS) tanks. Subsequently, maturation status (gonadosomatic index GSI and plasma levels of testosterone T and 17ß-estradiol E2) was assessed in one tank for each condition in January, February and during spawning in early April, while fish in the other tank were not disturbed in achieving reproductive success. Quantitative real-time PCR was performed to determine species-specific gonadotropin mRNA expression in females. Successful G1 spawning and egg fertilisation occurred in all experimental tanks. Gonadal development was similar under both conditions. Higher E2 and T levels were found in indoor housed females. Gonadotropin expression revealed similar profiles between outdoor and indoor housed females. G1 sole could be reproduced in the outdoor tanks under the natural photoperiod and in the indoor tanks under artificial simulation of this regime that includes a potentially crucial chilling period of 2-3 months at 5-7 °C.

Quantitative bioassays for measuring biologically functional gonadotropins based on eel gonadotropic receptors.

Significant declines in eel stocks have been noted in many parts of the world. Because eel aquaculture is dependent on wild-caught juveniles, there is a need to achieve artificial reproduction. Adult eel maturation is currently induced by repeated injections of purified gonadotropin (human chorionic gonadotropin [hCG]) or pituitary extract. Thus the determination of the biological efficacy and quantification of internal levels of gonadotropic hormones is important for optimizing artificial reproduction protocols. To quantify the plasma levels of biologically functional gonadotropic hormones, we developed a bioassay for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) based on the stable expression of receptors in HEK293 cells of the Japanese eel Anguilla japonica LH (ajLHR) and the European eel Anguilla anguilla FSH (aaFSHR), respectively. Such cells also contain a firefly luciferase reporter gene driven by a cAMP-responsive element (CRE-Luc). We found that the obtained stable cells, with ajLHR, responded linearly to a more than 100,000-fold concentration range of hCG diluted in saline. The cells with aaFSHR showed a linear response to a 1000-fold concentration range of salmon pituitary extract mixed with saline. The biological functionality of the LH and FSH bioassays was validated using hCG, human FSH, and pituitary extracts from salmon, carp and eel. Since the toxins in eel plasma damaged the HEK293 cells, the protocol was adapted to selectively inactivate the toxins by heating at 37°C for 24h. This process successfully enabled the monitoring of hormone levels in blood plasma sampled from hCG-injected eels. In this paper, we describe the development of gonadotropin bioassays that will be useful for improving reproduction protocols in eel aquaculture.

Models for the study of tendinopathy.

Tendinopathy refers to the clinical presentation of activity-related pain, focal tendon tenderness, and intratendinous imaging changes. The underlying pathology was once thought to be due to inflammation ('tendinitis'), but is now considered to predominantly result from degeneration ('tendinosis'). While some progress has been made in understanding tendinosis, the condition remains poorly understood and a need exists for suitable exploratory preclinical models. It is unlikely that one suitable model exists because of the complexity of the underlying pathology and myriad of possible causes. This paper provides an overview of current models utilized in tendinopathy research. It progresses hierarchically from in vitro and ex vivo models to in vivo models. For each model, rationale for use, pertinent findings, and advantages and disadvantages are discussed. By improving on these models, new methods for the prevention and treatment of tendinopathy may be explored with the ultimate outcome being a reduction in the occurrence and effects of the condition in humans.

Functional reorganization of cervical flexor activity because of induced muscle pain evaluated by muscle functional magnetic resonance imaging.

There is mounting evidence of an association between chronic neck pain and impaired cervical flexor muscle performance. It is likely that the deep cervical flexors demonstrate changes very early after the onset of pain, but evidence is currently lacking. This study investigated the effect of experimental neck muscle pain on the activation of the cervical flexor muscles during the performance of craniocervical flexion (CCF) by use of muscle functional magnetic resonance imaging. Activity of the longus colli (Lco), longus capitis (Lca) and sternocleidomastoid (SCM) muscles were investigated bilaterally and at three cervical levels (C0-C1, C2-C3 and C6-C7) in 14 healthy subjects. Measurements were performed at rest and after the performance of CCF without and with induced pain of the upper trapezius (intramuscular injection of hypertonic saline). In the non-pain condition, the Lca (p = 0.005) and Lco (p = 0.029) were significantly more active during CCF compared to SCM. In the pain condition, the activity of the Lco and Lca was reduced bilaterally and at multiple levels (p ≤ 0.009), whereas the left SCM showed increased activity at only the C6-C7 level (p ≤ 0.001). The results suggest that local excitation of nociceptive afferents causes an immediate reorganization of the cervical flexor muscle activity similar to that identified in clinical populations.

Is single-dose NVP relevant in the era of more efficacious PMTCT regimens? Lessons from Zambia.

For almost a decade, single-dose nevirapine (sdNVP) has been proven to be a safe and effective drug for the prevention of mother-to-child transmission (PMTCT) of HIV. With the advent of the use of more efficacious combination therapy strategy in reducing mother-to-child transmission, sdNVP has been relegated as a lower tier intervention. Availability of infrastructural capacity coupled with the practical reality that very few women attend an antenatal clinic more than once makes universal implementation of combination therapy a challenge. This retrospective review examined PMTCT programmatic indicators following the introduction of sdNVP at first contact in selected sites. Data from 79 PMTCT sites was reviewed from April 2006 to March 2007 (when sdNVP was offered only after 32 weeks) and compared to the period of April 2007-March 2008. In the pre-intervention period (April 2006-March 2007), the monthly average of pregnant women who received sdNVP per site was 5.02. Post-intervention (April 2007-March 2008), the monthly average increased by 59% to 7.97 (p-value<0.05). In pre-intervention period when sdNVP was dispensed at 32 weeks, the average proportion of pregnant women who received antiretroviral prophylaxis was 59%. This increased to 82% after the intervention. Current systems for dispensing sdNVP may be used as a foundation for implementation of more efficacious PMTCT regimens. The sdNVP administered at first contact should be a safety net for women who are unable to receive more efficacious regimen.

Alignment of the cell nucleus from labeled proteins only for 4D in vivo imaging.

Studies of protein dynamics by 4D (3D + time) confocal microscopy in vivo are hampered by global cell motion. The time between the acquisitions of the 3D images is in the order of minutes. Therefore, it is not to be expected that the cell as a whole remains fixed in the water basin on the stage. This superimposes a motion on the protein dynamics that has to be removed. We present a robust registration technique to align the cell images that does not require the a priori establishment of point-to-point correspondences. Instead, it uses the distribution of the labeled proteins. After correction for the translation, the 3D rotation of the cell is estimated. A robust intrinsic body coordinate system is constructed via the inertia tensor from the intensity distribution. By combining basis transformation to this intrinsic coordinate system, we can calculated the rotation matrix in a conceptual and computational straightforward manner. We have evaluated the performance of this approach in three experiments with human osteaosarcoma cells (U-2 OS), where the nuclear proteins Histon H4 and PML were visualized. The PML is concentrated in several dozen nuclear spots. Expression of Histon H4 results in a total nuclear staining. The registration results for both channels computed independently are very similar. Practically, this means that only the labeled material needs to be observed and still registration of the cell as a whole can be achieved.

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.

A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction.

Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogeneous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology.

Novel fibroblast growth factor 2 transcripts are expressed in mouse embryos.

We have discovered two new exons in the mouse fibroblast growth factor 2 (FGF-2 or bFGF) gene that can be alternatively spliced to the second coding exon of the gene. The newly identified exons 1b and 1c are located at, respectively, approximately 19 and 32 kb downstream of the canonical exon 1a. Using RT-PCR analysis, mRNAs containing exon 1c and canonical exons 2 and 3 were identified in embryonic limb, placenta, face, carcass and ocular tissues. A 3.7-kb transcript present in placenta and embryonic limb hybridizes with an exon 1c-derived probe in Northern blot analysis. Alternative splicing of exon 1c to exon 2 creates a transcript for which the predicted alternative FGF-2 (altFGF-2) polypeptide contains a novel N-terminal domain. Our data indicate that in mouse embryos multiple novel mRNA variants are transcribed from the FGF-2 locus using alternative splicing. These data suggest that proteins arising from these alternative transcripts may play a role in mouse embryogenesis.

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells.

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

African American dietary patterns at the beginning of the 20th century.

Early field studies in human nutrition documented the eating habits of African Americans living in a variety of circumstances. We compare the results of these investigations. Our analysis shows systematic differences along a continuum reaching from remote, rural communities in the South toward increasingly metropolitan locations. On the latter end of the continuum, we find diets richer in protein, composed of a wider variety of foods and containing fewer of what we now call "soul foods." Greater market involvement and access to low cost alternatives to more traditional foods help explain these developments.

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR.

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA(Lys) mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

Methods for visualizing RNA processing and transport pathways in living cells.

Recent advances in fluorescence microscopy, imaging, and probe technology provided possibilities to study the spatial and temporal distribution of RNA species in living cells. While some methods have been developed to localize all nascent or poly (A) containing transcripts others have been developed to study the in vivo distribution of specific RNA species. Irrespective of the method that has been used, the results of these studies provided important information concerning the localization and the cellular transport pathways of RNAs. Also, the picture emerges that RNA molecules travel through the nucleus at much faster speed, equaling that of free diffusion, than previously anticipated. Still, a major challenge proves to be the development of a microscopic detection technique that allows specific, in vivo, detection of low levels of RNA species by fluorescence in situ hybridization, without interfering fluorescent background signals derived from non-hybridized probe sequences and autofluorescent cell components. By applying photoactivatable caged fluorochrome-, molecular beacon-, or fluorescence resonance energy transfer (FRET)-based detection methods an important step in the future of living cell analysis has already been made.

Combination DNA/RNA FISH and immunophenotyping.

This unit presents methods for combining immunophenotyping with DNA/RNA FISH. The approach is used in so-called genotype/phenotype analysis to identify chromosomal aberrations in sub-populations of cells present in heterogenous populations. Combining RNA and DNA detection with identification of cellular proteins is quite difficult. This series of protocols is provided to enable the successful application of the combination of these techniques.

Mutational analysis of fibrillarin and its mobility in living human cells.

Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant approximately 0.02 microm(2) s(-1)), and has a significantly larger mobile fraction in CBs than in nucleoli.

Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome.

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.