RNA-FISH Technique: Probe Cocktails vs Single Probes

RNA-Fluorescence In Situ Hybridization (RNA-FISH) enables to analyze and visualize the microorganisms of interest within microbial communities in their natural environments by fluorescent labelling of specific RNA sequences. Poor accessibility or low content of the RNA target region can cause false positives/negatives due to low fluorescence of the cells. To reduce the chances of this occurring, probe cocktails – i.e. mixture of several probes that hybridize to different regions of the target RNA- has been proposed as an alternative to single probes use for increasing the Fluorescence Intensities (FI). However, is this really a good solution? The key finding of this work was that the use of probe cocktails is not always a good solution for maximizing the FI as at high concentrations the single probe EUK516-6 FAM yielded higher FI than the probe cocktails.

A SHORTENED RNA-FISH PROTOCOL FOR ANALYZING ARTWORKS’ MICROCOLONIZERS

Microorganisms are involved in the deterioration of Cultural Heritage. Thus, there is a need to enhance the techniques used for their detection and identification. RNA Fluorescent In Situ Hybridization (RNA-FISH) has been successfully applied for phylogenetic identification of the viable components of the microbial communities colonizing artworks both in situ and ex situ. Until recently, it was time-consuming, taking not less than 6 h for the analysis. We have developed an RNA-FISH in suspension protocol that allowed ex situ analysis of microorganisms involved in artworks’ biodeterioration in 5 h. In this work, three modified protocols, involving microwave heating, were evaluated for further shortening two of the four main critical steps in RNA-FISH: hybridization and washing. The original and modified protocols were applied in cellular suspensions of bacteria and yeast isolates. The results obtained were evaluated and compared in terms of detectability and specificity of the signals detected by epifluorescence microscopy. One of the methods tested showed good and specific FISH signals for all the microorganisms selected and did not produce signals evidencing non-specific or fixation-induced fluorescence. This 3 h protocol allows a remarkable reduction of the time usually required for performing RNA-FISH analysis in Cultural Heritage samples. Thus, a rapid alternative for analyzing yeast and bacteria cells colonizing artworks’ surfaces by RNA-FISH is presented in this work.

DETECTION OF SPORES AND HYPHAE OF ARTWORKS’ BIODETERIOGENIC FILAMENTOUS FUNGI BY RNA-FISH

Filamentous fungi are a threat to the conservation of Cultural Heritage. Thus, detection and identification of viable filamentous fungi are crucial for applying adequate Safeguard measures. RNA-FISH protocols have been previously applied with this aim in Cultural Heritage samples. However, only hyphae detection was reported in the literature, even if spores and conidia are not only a potential risk to Cultural Heritage but can also be harmful for the health of visitors, curators and restorers. Thus, the aim of this work was to evaluate various permeabilizing strategies for their application in the detection of spores/conidia and hyphae of artworks’ biodeteriogenic filamentous fungi by RNA-FISH. Besides of this, the influence of cell aging on the success of the technique and on the development of fungal autofluorescence (that could hamper the RNA-FISH signal detection) were also investigated. Five common biodeteriogenic filamentous fungi species isolated from biodegradated artworks were used as biological model: Aspergillus niger, Cladosporium sp, Fusarium sp, Penicillium sp. and Exophialia sp. Fungal autofluorescence was only detected in cells harvested from Fusarium sp, and Exophialia sp. old cultures, being aging-dependent. However, it was weak enough to allow autofluorescence/RNA-FISH signals distinction. Thus, autofluorescence was not a limitation for the application of RNA-FISH for detection of the taxa investigated. All the permeabilization strategies tested allowed to detect fungal cells from young cultures by RNA-FISH. However, only the combination of paraformaldehyde with Triton X-100 allowed the detection of conidia/spores and hyphae of old filamentous fungi. All the permeabilization strategies failed in the Aspergillus niger conidia/spores staining, which are known to be particularly difficult to permeabilize. But, even in spite of this, the application of this permeabilization method increased the analytical potential of RNA FISH in Cultural Heritage biodeterioration. Whereas much work is required to validate this RNA-FISH approach for its application in real samples from Cultural Heritage it could represent an important advance for the detection, not only of hyphae but also of spores and conidia of various filamentous fungi taxa by RNA-FISH.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization

An unusual feature of the mammalian genome is the number of genes exhibiting monoallelic expression. Recently random monoallelic expression of autosomal genes has been reported for olfactory and Ly-49 NK receptor genes, as well as for Il-2, Il-4 and Pax5. RNA fluorescence in situ hybridization (FISH) has been exploited to monitor allelic expression by visualizing the number of sites of transcription in individual nuclei. However, the sensitivity of this technique is difficult to determine for a given gene. We show that by combining DNA and RNA FISH it is possible to control for the hybridization efficiency and the accessibility and visibility of fluorescent probes within the nucleus.

Novos desenvolvimentos para a deteção de leveduras e bactérias presentes em argamassas

The development of simple and rapid new approaches for analysing microbial communities colonising Cultural Heritage materials is pivotal for its safeguard. Fluorescence in situ hybridisation technique using ribosomal RNA directed probes (RNA-FISH) has demonstrated a great potential for this purpose. A protocol for analysing filamentous fungi in mortars has been already developed in previous studies. In this work this protocol has been adapted for detecting bacteria and yeasts. Good results have been obtained for the analysis of suspensions of isolates. In this way, the optimized protocol was applied in microsamples from synthetic mortar artificially inoculated with yeast and bacterial isolates. Promising results have been obtained for the ex situ analysis of yeast and bacteria thriving in mortar microsamples.

Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus

In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.

Conserved gene structure and function of interleukin-10 in teleost fish

Interleukin-10 (IL-10) is an important immunoregulatory cytokine produced by various types of cells. Researchers describe here the isolation and characterization of olive flounder IL-10 (ofIL-10) cDNA and genomic organization. The ofIL-10 gene encodes a 187 amino acid protein and is composed of a five exon/four intron structure, similar to other known IL-10 genes. The ofIL-10 promoter sequence analysis shows a high level of homology in putative binding sites for transcription factors which are sufficient for transcriptional regulation ofIL-10. Important structural residues are maintained in the ofIL-10 protein including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10 and two extra cysteine residues that exist only in fish species. The phylogenetic analysis clustered ofIL-10 with other fish IL-10s and apart from mammalian IL-10 molecules. Quantitative real-time Polymerase Chain Reaction (PCR) analysis demonstrated ubiquitous ofIL-10 gene expression in the 13 tissues examined. Additionally, the induction of ofIL-10 gene expression was observed in the kidney tissue from olive flounder infected with bacteria (Edawardsiella tarda) or virus (Viral Hemorrhagic Septicemia Virus; VHSV). These data indicate that IL-10 is an important immune regulator that is conserved strictly genomic organization and function during the evolution of vertebrate immunity.

Evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypophthalmus) using an RNA-Seq approach

Increasing salinity levels in freshwater and coastal environments caused by sea level rise linked to climate change is now recognized to be a major factor that can impact fish growth negatively, especially for freshwater teleost species. Striped catfish (Pangasianodon hypophthalmus) is an important freshwater teleost that is now widely farmed across the Mekong River Delta in Vietnam. Understanding the basis for tolerance and adaptation to raised environmental salinity conditions can assist the regional culture industry to mitigate predicted impacts of climate change across this region. Attempt of next generation sequencing using the ion proton platform results in more than 174 million raw reads from three tissue libraries (gill, kidney and intestine). Reads were filtered and de novo assembled using a variety of assemblers and then clustered together to generate a combined reference transcriptome. Downstream analysis resulted in a final reference transcriptome that contained 60,585 transcripts with an N50 of 683 bp. This resource was further annotated using a variety of bioinformatics databases, followed by differential gene expression analysis that resulted in 3062 transcripts that were differentially expressed in catfish samples raised under two experimental conditions (0 and 15 ppt). A number of transcripts with a potential role in salinity tolerance were then classified into six different functional gene categories based on their gene ontology assignments. These included; energy metabolism, ion transportation, detoxification, signal transduction, structural organization and detoxification. Finally, we combined the data on functional salinity tolerance genes into a hypothetical schematic model that attempted to describe potential relationships and interactions among target genes to explain the molecular pathways that control adaptive salinity responses in P. hypophthalmus. Our results indicate that P. hypophthalmus exhibit predictable plastic regulatory responses to elevated salinity by means of characteristic gene expression patterns, providing numerous candidate genes for future investigations.

Isolation of transforming growth factor-beta 2 cDNA from a fish, Cyprinus carpio by RT-PCR

Transforming Growth Factors-beta (TGF-beta s) have been described in many vertebrate species of amphibians, aves and mammals. In this report we demonstrate the presence of TGF-beta 2 in pisces. TGF-beta 2 has been cloned from a fish, Cyrinus carpio, by RT-PCR using degenerate oligonucleotide primers. Sequence analysis of the amplified product and alignment of the deduced amino acid sequence with the human TGF-beta 2 amino acid sequence revealed 81% and 93% identity in the precursor and the mature regions, respectively. The northern blot analysis of fish heart RNA shows a major messenger RNA species of about 8.0 kb and two messages of very low abundance of about 5.0 kb and 4.0 kb. The identification of TGF-beta 2 isoform in Pisces and it's high degree of homology with the mammalian isoform suggests that among all TGF-beta isoforms, TGF-beta 2 is the most conserved during evolution. (C) 1997 Elsevier Science B.V.

Mugilid Fish Are Sentinels of Exposure to Endocrine Disrupting Compounds in Coastal and Estuarine Environments

Effects on fish reproduction can result from a variety of toxicity mechanisms first operating at the molecular level. Notably, the presence in the environment of some compounds termed endocrine disrupting chemicals (EDCs) can cause adverse effects on reproduction by interfering with the endocrine system. In some cases, exposure to EDCs leads to the animal feminization and male fish may develop oocytes in testis (intersex condition). Mugilid fish are well suited sentinel organisms to study the effects of reproductive EDCs in the monitoring of estuarine/marine environments. Up-regulation of aromatases and vitellogenins in males and juveniles and the presence of intersex individuals have been described in a wide array of mullet species worldwide. There is a need to develop new molecular markers to identify early feminization responses and intersex condition in fish populations, studying mechanisms that regulate gonad differentiation under exposure to xenoestrogens. Interestingly, an electrophoresis of gonad RNA, shows a strong expression of 5S rRNA in oocytes, indicating the potential of 5S rRNA and its regulating proteins to become useful molecular makers of oocyte presence in testis. Therefore, the use of these oocyte markers to sex and identify intersex mullets could constitute powerful molecular biomarkers to assess xenoestrogenicity in field conditions.

Identification of Sex and Female’s Reproductive Stage in Commercial Fish Species through the Quantification of Ribosomal Transcripts in Gonads

The estimation of maturity and sex of fish stocks in European waters is a requirement of the EU Data Collection Framework as part of the policy to improve fisheries management. On the other hand, research on fish biology is increasingly focused in molecular approaches, researchers needing correct identification of fish sex and reproductive stage without necessarily having in house the histological know-how necessary for the task. Taking advantage of the differential gene transcription occurring during fish sex differentiation and gametogenesis, the utility of 5S ribosomal RNA (5S rRNA) and General transcription factor IIIA (gtf3a) in the molecular identification of sex and gametogenic stage was tested in different economically-relevant fish species from the Bay of Biscay. Gonads of 9 fish species (, Atlantic, Atlantic-chub and horse mackerel, blue whiting, bogue, European anchovy, hake and pilchard and megrim), collected from local commercial fishing vessels were histologically sexed and 5S and 18S rRNA concentrations were quantified by capillary electrophoresis to calculate a 5S/18S rRNA index. Degenerate primers permitted cloning and sequencing of gtf3a fragments in 7 of the studied species. 5S rRNA and gtf3a transcript levels, together with 5S/18S rRNA index, distinguished clearly ovaries from testis in all of the studied species. The values were always higher in females than in males. 5S/18S rRNA index values in females were always highest when fish were captured in early phases of ovary development whilst, in later vitellogenic stages, the values decreased significantly. In megrim and European anchovy, where gonads in different oogenesis stages were obtained, the 5S/18S rRNA index identified clearly gametogenic stage. This approach, to the sexing and the quantitative non-subjective identification of the maturity stage of female fish, could have multiple applications in the study of fish stock dynamics, fish reproduction and fecundity and fish biology in general.

Estimation of RNA/DNA ratio in two north-Indian natural populations of mahseer (Tor tor) and its relationship with growth and hydrobiology

Samples of Tor tor were collected from Bari Reservoir of Udaipur and Narmada River at Hoshangabad (India), in the months of July and November 2005, respectively. Twenty-five samples were collected from each location. Bari Reservoir samples ranged from 17.0 to 24.5 cm in total length and from 75 to 155 g in weight, while Narmada samples ranged from 20.0 to 42.0 cm in length and 90 to 425 g in weight. The nucleic acid content in body muscle of Tor tor and the RNA/DNA ratio were estimated. The age of fishes was estimated by the scale study method and specimens were classified into four age groups. RNA/DNA ratio showed significant linear increase with increase in weight and age till the age of three years after which, the growth rate reduced. The 1-2 year group was the only one common between the two water bodies and a comparison of RNA/DNA ratios showed higher growth rate in Bari Reservoir. The gross primary productivity was also higher in Bari Reservoir being 551 mg cmˉ³ dˉ¹ compared to 404 mg cmˉ³ dˉ¹ observed for Narmada River. The condition factor (K) was found to be higher (1.21) in the fish from the Bari Reservoir compared to those of Narmada River (1.14). The growth rate was higher in females compared to males in >100 g specimens.

Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .

Isolation and characterization of Argonaute 2: A key gene of the RNA interference pathway in the rare minnow, Gobiocypris rarus

Argonaute 2 gene plays a pivotal role in RNAi in many species. Herein is the first report of the cloning and characterization of Argonaute 2 gene in fish. The full-length cDNA of Gobiocypris rarus Argonaute 2 (GrAgo2) consisted of 3073 nucleotides encoding 869 amino acid residues with a calculated molecular weight of 98.499 kDa and an estimated isoelectric point of 9.18. Analysis of the deduced amino acid sequence showed the presence of two signature domains, PAZ and Piwi. RT-PCR analysis indicated that GrAgo2 mRNA expression could be detected in widespread tissues. After infection with grass carp reovirus, GrAgo2 expression was up-regulated from 12 h post-injection (p < 0.05) and returned to control levels at 48 h post-injection (p > 0.05). These data imply that GrAgo2 is involved in antiviral defense in rare minnow. (C) 2008 Published by Elsevier Ltd.