Transcriptome analysis of buffalo granulosa cells in three dimensional culture systems.

Hanging drop (HD) three-dimensional (3D) culture model for buffalo granulosa cells (GC) was reported to mimic the preovulatory stage of ovarian follicles in our previous study. To further verify its reliability, the present study attempted a comparative transcriptome profile of buffalo GC freshly isolated from ovarian follicles (<8 mm diameter) (FC) and their cultures in normal culture dish (ND or 2D), polyHEMA coated dish (PH) and HD culture systems (3D). Out of 223 significantly (-log2 fold change: >3; p < .0005; false discovery rate [FDR]: <0.1) differentially expressed genes (SDEGs) among different culture systems, 137 were found unannotated, and 94, 29, and 66 were exclusively expressed in FC, PH, and HD, respectively. However, on eliminating the fixed points of p values and FDR from the entire raw data, only 11 genes related to long noncoding RNA, 12 genes related to luteinization, and 3 genes related to follicular maturation were exclusively expressed in FC, PH, and HD culture systems, respectively. The quantitative real time-PCR validation and the next generation sequencing data had more than 90% correlation. Bioinformatics analyses of the exclusively expressed SDEG revealed that the freshly aspirated GCs were a true representative of GCs from small follicles (<8 mm diameter), the GC spheroids under PH maintained mitochondrial function, and those cultured in HD system for 6 days simulated the inflammatory milieu required for ovulation. Therefore, the comparative transcriptome profile also reinforced that HD culture system is better in vitro culture method than the other methods analyzed in this study for buffalo GC.

Against All Odds: RNA Extraction From Different Protocols Adapted to Formalin-fixed Paraffin-embedded Tissue.

For the preservation of tissue samples, formalin fixation followed by paraffin embedding (FFPE) has been the method of choice for decades, mainly because it maintains the morphologic characteristics of the original tissue particularly preserved, as well as its genetic material. FFPE cells can be used to perform molecular tests, such as conventional (c) or quantitative (q) reverse transcriptase polymerase chain reaction (RT-PCR), in retrospective investigations. However, extracting RNA from archived FFPE tissues is a challenging procedure, as it requires time and the use of complex extraction methods. As specific FFPE extraction methods are not always available in the laboratories, the objective of this study was to evaluate the performance of a method based on phenol-chloroform (PC) and 2 commercial methods for RNA extraction, adapting their protocols for FFPE tissues. For this study, a pool of FFPE tissues underwent RNA extraction by PC, QIAmp Viral RNA Mini, and RNeasy Mini Kit. Both the RT-cPCR and the RT-qPCR results were favorable, demonstrating the viability of the RNA. As these results expanded the alternatives for low-budget FFPE extraction, the choice of the ideal method to be used will depend on the availability of reagents and kits.

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of toxigenic Fusarium temperatum in maize stalks and kernels.

Fusarium temperatum is an emerging maize pathogen that causes maize ear and stalk rot diseases and produces various mycotoxins including moniliformin, beauvericin, enniatins and fumonisin B1, which poses a potential risk to the human food or animal feed supply chains. Early detection of F. temperatum is crucial to prevent its derived mycotoxins from entering the food chain, and is also a useful tool in disease management practices. Here, we describe a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of F. temperatum. The 28S ribosomal DNA sequences (28S rDNA) of F. temperatum were used to design a set of six primers. The reaction conditions were optimized for developing a fast assay with high specificity and sensitivity, and were able to detect the presence of less than 10 pg of target DNA per reaction within 60 min. Furthermore, the resulting amplicons were visualized by adding SYBR Green I to the reaction tubes. Suspected F. temperatum infected maize stalk samples collected from Yunnan province, China were identified using the developed LAMP assay. In conclusion, the method not only provides a rapid and specific screening for the existence of F. temperatum in a bulk of maize samples without using sophisticated equipment, but also is potentially useful for other agriculturally important toxigenic fungi.

A multilocus molecular phylogeny of Fasciolariidae (Neogastropoda: Buccinoidea).

The neogastropod family Fasciolariidae Gray, 1853 - tulips, horse-conchs, spindles, etc., comprises important representatives of tropical and subtropical molluscan assemblages, with over 500 species in the subfamilies Fasciolariinae Gray, 1853, Fusininae Wrigley, 1927 and Peristerniinae Tryon, 1880. Fasciolariids have had a rather complicated taxonomical history, with several genus names for a long time used as waste baskets to group many unrelated species; based on shell characters, recent taxonomic revisions have, however, began to set some order in its taxonomy. The present work is the first molecular approach to the phylogeny of Fasciolariidae based on a multigene dataset, which provides support for fasciolariids, an old group with a fossil record dating back to the Cretaceous. Molecular markers used were the mitochondrial genes 16S rRNA and cytochrome c oxidase subunit I, and the nuclear genes 18S rRNA, 28S rRNA and histone H3, sequenced for up to 116 ingroup taxa and 17 outgroups. Phylogenetic analyses revealed monophyly of Dolicholatirus Bellardi, 1884 and Teralatirus Coomans, 1965, however it was not possible to discern if the group is the sister clade to the remaining fasciolariids; the latter, on the other hand, proved monophyletic and contained highly supported groups. A first split grouped fusinines and Pseudolatirus Bellardi, 1884; a second split grouped the peristerniine genera Peristernia Mörch, 1852 and Fusolatirus Kuroda and Habe, 1971, while the last group comprised fasciolariines and the remaining peristerniines. None of these clades correspond to the present-day accepted circumscription of the three recognized subfamilies.

The phylogeny and systematics of the Nassariidae revisited (Gastropoda, Buccinoidea).

Nassariidae are a group of scavenging, predominantly marine, snails that are diversified on soft bottoms as well as on rocky shores, and are the subject of numerous research papers in ecology, ecotoxicology or paleontology. A weak and/or apparently continuous variation in shell characters has resulted in an intimidating taxonomy, with complex synonymy lists. Over 1320 extant nominal species have been described, of which 442 are currently regarded as valid. Above species level, the state of the art is equally hazy, with four subfamilies and twelve genera currently accepted, and many other names in the graveyard of synonymy. A molecular analysis based on three mitochondrial (COI, 16S, 12S) and two nuclear (28S, H3) markers was conducted. Our dataset includes 218 putative nassariid species, comprising 9 of the 12 valid genera, and 25 nominal genera represented by their type species. The monophyly of the Nassariidae as classically construed is not confirmed. Species of Antillophos, Engoniophos, Phos, Nassaria, Tomlinia and Anentome (formerly considered Buccinidae) are included inside the Nassariidae clade. Within the Nassariinae, the tree unexpectedly demonstrates that species from the Atlantic and the Indo-Pacific form different clades which represent several independent diversification events. Through an integrative approach, the reconstruction of ancestral states was addressed for eight characters supposedly informative for taxonomy. Using numerous fossil calibration points, Nassariidae appear to have originated 120 MYA ago in Atlantic temperate waters during the Lower Cretaceous. Our results have a profound impact on nassariid taxonomy, especially with regard to the validity of subfamily- and genus-level names.

A comprehensive molecular phylogeny of spoon worms (Echiura, Annelida): Implications for morphological evolution, the origin of dwarf males, and habitat shifts.

Echiurans (spoon worms) are derived annelids that have secondarily lost segmentation. Recently, two molecular phylogenetic studies were performed to resolve the interfamily relationship of echiurans. However, the tree topologies were incongruent and taxon sampling was limited in both the studies. Thus, the phylogenetic relationships within echiurans remain contentious. In this study, I reevaluated the molecular phylogeny of echiurans, using three nuclear (18S, 28S, and H3) and two mitochondrial (16S and COI) genes of 49 echiuran species belonging to 17-19 genera and five families. Results showed that echiurans form the following two major clades: a sexually monomorphic group (Echiuridae, Urechidae, and Thalassematidae) and a sexually dimorphic group (Bonelliidae and Ikedidae). The sister group relationships between Urechidae and Echiuridae, as well as between Ikedidae and Bonelliidae, were supported. The analysis also supported the following relationships among genera within Thalassematidae: {Arhynchite [(Thalassema, Lissomyema) (Ochetostoma, Listriolobus, Ikedosoma, Anelassorhynchus)]}. Furthermore, I evaluated the evolutionary patterns of important taxonomic characteristics (body-wall longitudinal musculature, proboscis shape, gonostmal lip shape, and body color) and habitat shifts (water depth and substrate type), using ancestral state reconstruction analyses. The analyses showed that sexually dimorphic echiurans originated in the shallow waters and secondarily invaded the deep sea, although deep-to-shallow habitat reversal was also detected. In contrary to the previous hypothesis, sexual dimorphism with dwarf males in echiurans may have been a preadaptation to the deep-sea environment. The analyses also showed that habitat shifts from soft sediments to hard substrates occurred in Thalassematidae and Bonelliidae, respectively. A new classification of echiurans, in which Echiura comprises two superfamilies, namely Echiuroidea (with Echiuridae, Urechidae, and Thalassematidae) and Bonellioidea (with Bonelliidae and Ikedidae), is proposed.

Detection of tropical fungi in formalin-fixed, paraffin-embedded tissue: still an indication for microscopy in times of sequence-based diagnosis?

INTRODUCTION: The aim of the study was the evaluation of panfungal PCR protocols with subsequent sequence analysis for the diagnostic identification of invasive mycoses in formalin-fixed, paraffin-embedded tissue samples with rare tropical mycoses. MATERIALS AND METHODS: Five different previously described panfungal PCR/sequencing protocols targeting 18S and 28S ribosomal RNA gene fragments as well as internal transcribed spacer 1 and 2 fragments were evaluated with a collection of 17 formalin-fixed, paraffin-embedded tissue samples of patients with rare and/or tropical invasive mycoses, comprising chromoblastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, mycetoma/maduromycosis, and rhinosporidiosis, in a proof-of-principle analysis. RESULTS: The primers of the panfungal PCRs readily and predominantly reacted with contaminating environmental fungi that had deposited on the paraffin blocks. Altogether three sequence results of histoplasmosis and mycetoma samples that matched the histological assessment were associated with sample age <10 years and virtually without PCR inhibition. CONCLUSIONS: The high risk of amplifying environmental contaminants severely reduces the usefulness of the assessed panfungal PCR/sequencing protocols for the identification of rare and/or tropical mycoses in stored formalin-fixed, paraffin-embedded tissues. Histological assessment remains valuable for such indications if cultural differentiation is impossible from inactivated sample material.

Karyotype evolution in monitor lizards: cross-species chromosome mapping of cDNA reveals highly conserved synteny and gene order in the Toxicofera clade.

The water monitor lizard (Varanus salvator macromaculatus (VSA), Platynota) has a chromosome number of 2n = 40: its karyotype consists of 16 macrochromosomes and 24 microchromosomes. To delineate the process of karyotype evolution in V. salvator macromaculatus, we constructed a cytogenetic map with 86 functional genes and compared it with those of the butterfly lizard (Leiolepis reevesii rubritaeniata (LRE); 2n = 36) and Japanese four-striped rat snake (Elaphe quadrivirgata (EQU); 2n = 36), members of the Toxicofera clade. The syntenies and gene orders of macrochromosomes were highly conserved between these species except for several chromosomal rearrangements: eight pairs of VSA macrochromosomes and/or chromosome arms exhibited homology with six pairs of LRE macrochromosomes and eight pairs of EQU macrochromosomes. Furthermore, the genes mapped to microchromosomes of three species were all located on chicken microchromosomes or chromosome 4p. No reciprocal translocations were found in the species, and their karyotypic differences were caused by: low frequencies of interchromosomal rearrangements, such as tandem fusions, or centric fissions/fusions between macrochromosomes and between macro- and microchromosomes; and intrachromosomal rearrangements, such as paracentric inversions or centromere repositioning. The chromosomal rearrangements that occurred in macrochromosomes of the Varanus lineage were also identified through comparative cytogenetic mapping of V. salvator macromaculatus and V. exanthematicus. Morphologic differences in chromosomes 6-8 between the two species could have resulted from pericentric inversion or centromere repositioning.

Fasciola hepatica - where is 28S ribosomal RNA?

Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.

Isolation of high-quality RNA from stented blood vessels.

Stents have become a standard of care for the treatment of coronary artery disease. A series of cellular and molecular processes contribute to the vascular response following stent placement. For the purpose of local gene expression studies, metallic stent struts are usually removed from the vessel wall with forceps under a dissection microscope prior to RNA extraction. Main drawbacks of the manual dissection are that it may cause additional tissue damage and compromise the quality of RNA through prolonged tissue handling. In this technical note, we report the recovery of high-quality RNA from atherosclerotic vessels with stent struts left in situ.

Multiplex real-time PCR method for detection and quantification of mycotoxigenic fungi belonging to three different genera.

UNLABELLED: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real-time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene-producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON- and OTA-producing fungi can be detected and quantified in a single reaction tube using this multiplex real-time PCR method. PRACTICAL APPLICATION: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal-based foods.

Development of a single-step subtraction method for eukaryotic 18S and 28S ribonucleic acids.

The abundance of mammalian 18S and 28S ribosomal RNA can decrease the detection sensitivity of bacterial or viral targets in complex host-pathogen mixtures. A method to capture human RNA in a single step was developed and characterized to address this issue. For this purpose, capture probes were covalently attached to magnetic microbeads using a dendrimer linker and the solid phase was tested using rat thymus RNA (mammalian components) with Escherichia coli RNA (bacterial target) as a model system. Our results indicated that random capture probes demonstrated better performance than specific ones presumably by increasing the number of possible binding sites, and the use of a tetrame-thylammonium-chloride (TMA-Cl-) based buffer for the hybridization showed a beneficial effect in the selectivity. The subtraction efficiency determined through real-time RT-PCR revealed capture-efficiencies comparable with commercially available enrichment kits. The performance of the solid phase can be further fine tuned by modifying the annealing time and temperature.

A novel Sarcocystis-associated encephalitis and myositis in racing pigeons.

Sarcosporidian cysts in the skeletal muscle of domestic pigeons (Columba livia f. domestica) have previously been attributed to infection with Sarcocystis falcatula, which is shed in the faeces of the opossum (Didelphis virginiana). Here, we describe fatal spontaneous encephalitis and myositis associated with Sarcocystis infections in three flocks of racing pigeons with 47 of 244 animals affected. The clinical course was characterized by depression, mild diarrhoea, torticollis, opisthotonus, paralysis and trembling. Histopathological examination of 13 pigeons revealed generalized severe granulomatous and necrotizing meningoencephalitis and myositis with sarcosporidian cysts. Light and transmission electron microscopy identified cysts in heart and skeletal muscle of 1 to 2 mm in length and 20 to 50 microm in width. These were subdivided into small chambers by fine septae and filled with lancet-shaped cystozoites (7.5 x 1.5 microm) and dividing metrocytes, which is characteristic for Sarcocystis. The cysts had smooth walls and were devoid of protrusions typical of S. falcatula. Polymerase chain reaction amplification and sequencing of the internal transcribed spacer region (ITS-1) and the complete 28S rRNA identified a novel Sarcocystis species with only 51% ITS-1 nucleotide sequence similarity with S. falcatula. A phylogenetic comparison of the 28S rRNA revealed close sequence homologies with Frenkelia microti, Frenkelia glareoli and Sarcocystis neurona. The clinical, histopathological, electron microscopic and genetic data are unlike any previously described protozoan infections in pigeons, suggesting a novel, severe disease due to an as yet undescribed Sarcocystis species.

Ribosomal DNA sequence variation among sympatric strains of the Cyclotella meneghiniana complex (Bacillariophyceae) reveals cryptic diversity.

Cyclotella meneghiniana Kützing is one of the most commonly found and intensively studied freshwater diatom species. However, it is considered taxonomically problematic because of its unusually wide ecological range and large frustule ultrastructural variation. As part of a study of morphological and genetic variation in this morphospecies, we surveyed nucleotide variation in the hypervariable D1/D2 regions of the 28S rDNA, in the ribosomal internal transcribed spacer region (containing ITS1, the 5.8S rDNA and ITS2) and in the 18S rDNA in a collection of 20 sympatric strains. High genetic variability and strong indications of genetic structure among the Cyclotella meneghiniana strains were found. Representatives of four genetically distinct--apparently reproductively isolated--groups were revealed among them. The random distribution of ITS variation within these four groups indicated that the genetic structure in Cyclotella meneghiniana can probably be explained by the presence of cryptic sexual species rather than by the lack of allogamous sexual reproduction. The morphological features traditionally used for species identification in this group cannot distinguish these putative cryptic species.

Genetic relationships between blowflies (Calliphoridae) of forensic importance.

Phylogenetic relationships among blowfly (Calliphoridae) species of forensic importance are explored using DNA sequence data from the large sub-unit (lsu, 28S) ribosomal RNA (rRNA) gene, the study includes representatives of a range of calliphorid species commonly encountered in forensic analysis in Britain and Europe. The data presented provide a basis to define molecular markers, including the identification of highly informative intra-sequence regions, which may be of use in the identification of larvae for forensic entomology. Phylogenetic analysis of the sequences also provides new insights into the different evolutionary patterns apparent within the family Calliphoridae which, additionally, can provide a measure of the degree of genetic variation likely to be encountered within taxonomic groups of differing forensic utility.

Proposed secondary structure of eukaryote specific expansion segment 15 in 28S rRNA from mice, rats, and rabbits.

The expansion segments in eukaryotic ribosomal RNAs are additional RNA sequences not found in the RNA core common to both prokaryotes and eukaryotes. These regions show large species-dependent variations in sequence and size. This makes it difficult to create secondary structure models for the expansion segments exclusively based on phylogenetic sequence comparison. Here we have used a combination of experimental data and computational methods to generate secondary structure models for expansion segment 15 in 28S rRNA in mice, rats, and rabbits. The experimental data were collected using the structure sensitive reagents DMS, CMCT, kethoxal, micrococcal nuclease, RNase T(1), RNase CL3, RNase V(1), and lead(II) acetate. ES15 was folded with the computer program RNAStructure 3.5 using modification data and phylogenetic similarities between different ES15 sequences. This program uses energy minimization to find the most stable secondary structure of an RNA sequence. The presented secondary structure models include several common structural motifs, but they also have characteristics unique to each organism. Overall, the secondary structure models showed indications of an energetically stable but dynamic structure, easily accessible from the solution by the modification reagents, suggesting that the expansion segment is located on the ribosomal surface.

Effect of oxytocin on expression of cytosolic phospholipase A2 mRNA and protein in ovine endometrial tissue in vivo.

The induction of endometrial prostaglandin (PG) F2alpha synthesis by oxytocin is dependent upon activation of phospholipase (PL) A2 and mobilization of arachidonic acid. The objective of this study was to determine if oxytocin stimulates PGF2alpha synthesis by inducing synthesis of cytosolic PLA2 (cPLA2). In Experiment 1, 15 ovariectomized ewes were given progesterone and estradiol to simulate an estrous cycle. Ewes were then given an injection of oxytocin on Day 14 of the simulated estrous cycle. Jugular blood samples were collected and assayed for 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM). Uteri were collected at 0, 7.5, 25, 90, or 240 min postinjection (n = 3 ewes/time point). Total RNA was isolated from caruncular endometrium and subjected to dot-blot analysis. Oxytocin induced a rapid and transient increase in serum PGFM (P < 0.01). However, endometrial concentrations of cPLA2 mRNA did not change following oxytocin administration (P > 0.10). In Experiment 2, 11 ovary-intact ewes were given oxytocin (n = 5) or saline (n = 6) on Day 15 after estrus. Jugular blood samples were collected and assayed for serum concentrations of PGFM. Uteri were collected at 15 min postinjection. Homogenates were prepared from caruncular endometrium and subjected to Western blot analysis. Concentrations of PGFM were higher in oxytocin treated ewes compared to saline treated ewes at 15 min postinjection (P < 0.01). Endometrial concentrations of cPLA2 protein were greater in the cytosolic than in the microsomal fraction (P < 0.01). Oxytocin did not affect the amount of cPLA2 protein in either fraction (P > 0.10). In conclusion, oxytocin did not effect expression of either cPLA2 mRNA or protein in ovine endometrium. Oxytocin may stimulate PGF2alpha synthesis by activating cPLA2 protein that is already present in an inactive form.

Sequence comparison of avian interferon regulatory factors and identification of the avian CEC-32 cell as a quail cell line.

Interferon (IFN) regulatory factor-1 (IRF-1) is a well-characterized member of the IRF family. Previously, we have cloned cDNA of several members of the chicken IRF (ChIRF) family and studied the function of ChIRF-1 in the avian cell line CEC-32. The IRF-1 proteins from primary chicken embryo fibroblasts (CEF) and CEC-32 cells differed in their electrophoretic mobility. To characterize the different forms of IRF-1 in avian cells, we compared the sequences of IRF-1 cDNA from CEC-32 cells, primary CEF, and quail fibroblasts (QEF). The deduced amino acid sequences of IRF-1 cDNA from chicken and quail show high similarity. Comparison of genomic sequences of IRF-1 and IFN consensus sequence binding protein (ICSBP) also confirm the relatedness of the members of the IRF family in quail and chicken. Based on these data, it is concluded that the avian fibroblast cell line CEC-32 is derived from quail. This conclusion is further supported by deoxynucleotide sequence comparison of a DNA fragment in an avian MHC class II gene and by fluorescence in situ hybridization (FISH) using the vertebrate telomeric (TTAGGG) repeat. Chromosome morphology and the lack of interstitial hybridization signals in macrochromosomes suggest that the CEC-32 cell line has probably been derived from Japanese quail.

The 28S fraction of rRNA in molluscs displays electrophoretic behaviour different from that of mammal cells.

Electrophoresis on agarose/formaldehyde gels of rRNA in molluscs display a pattern of bands which could suggest a RNase action due to incorrect manipulation of the samples. This study shows that the disappearance of the band corresponding to the 28S fraction is due to the denaturing conditions used when electrophoresis is carried out and not to RNases action.