Relative Transcript Abundance Research Articles

Molecular characterization of the lipophorin receptor in the crustacean ectoparasite Lepeophtheirus salmonis.

The Salmon louse (Lepeophtheirus salmonis) is a marine ectoparasite of salmonid fish in the Northern Hemisphere and considered as a major challenge in aquaculture and a threat to wild populations of salmonids. Adult female lice produce a large number of lipid-rich eggs, however, the mechanism of maternal lipid transport into developing eggs during salmon louse reproduction has not been described. In the present study, a full-length L. salmonis lipophorin receptor (LsLpR) consisting of 16 exons was obtained by RACE and RT-PCR. The predicted ORF was 952 amino acids and structural analysis showed five functional domains that are similar to LpR of insects and decapods. Phylogenetic analysis placed the LsLpR together with LpRs from decapods and insects. Expression analysis revealed that the relative abundance of LsLpR transcripts was highest in the larvae and adult female lice. In adult females, the LsLpR transcripts and protein were found in the ovary and vitellogenic oocytes whereas, in larvae, the LsLpR transcripts were found in the neuronal somata of the brain and the intestine. Oil Red O stain results revealed that storage of neutral lipids was found in vitellogenic oocytes and ovaries of adult females, and in the yolk of larvae. Moreover, RNA interference (RNAi) was conducted to demonstrate the function of LsLpR in reproduction and lipid metabolism in L. salmonis. In larvae, the transcription of LsLpR was decreased by 44–54% while in an experiment LsLpR knockdown female lice produced 72% less offspring than control lice.

Effects of multiple life stage exposure to the fungicide prochloraz in Xenopus laevis: Manifestations of antiandrogenic and other modes of toxicity

The Larval Amphibian Growth and Development Assay (LAGDA) is an internationally harmonized testing guideline for evaluating effects of chronic chemical exposure in amphibians. In order to evaluate the effects of chronic exposure to an antiandrogenic chemical in an amphibian model, prochloraz was tested using a variation of the LAGDA design. Exposure was initiated with <1d post-fertilization embryos at nominal concentrations of 0, 6.7, 20, 60 and 180 μg/L (0, 18, 53, 159, 478 nM) and continued in flow-through conditions until two months following the median time that controls completed metamorphosis. Growth, developmental rate, circulating thyroid hormone and thyroid gland histopathology were evaluated in a subsample at completion of metamorphosis. There were no effects on growth or development at this stage, but circulating thyroid hormone was elevated in the 20, 60 and 180 μg/L treatments and minimal to mild thyroid follicular cell hypertrophy was observed histologically in the 180 μg/L treatment. Growth, overt toxicity, and reproductive development were evaluated at test termination. There were no effects on growth in either gender, but livers and kidneys exhibited treatment-related pathologies consistent with organ toxicity related to metabolism and presumably impaired excretion of prochloraz metabolites. Histological assessments of female ovaries resulted in minimal pathologies only in the 180 μg/L treatment while male testes exhibited numerous treatment-related pathologies that are consistent with previously reported antiandrogenic effects of prochloraz in other species. The most severe testis pathologies occurred in the 180 μg/L treatment; however, incidences of treatment-related pathologies occurred in all prochloraz treatments. Müllerian duct regression in males was inhibited by prochloraz exposure while Müllerian duct maturation in females was accelerated, characteristic of a feminizing effect. Gene expression levels of potential biomarkers of testis function were also measured. Relative abundance of cyp17a1 transcripts was generally unaffected by prochloraz exposure whereas the Insl3 orthologue, rflcii, was elevated by 3 and >5-fold in the 60 and 180 μg/L treatments, respectively, indicating impaired Leydig cell maturation and testosterone signaling. Overall, prochloraz exposure caused effects characteristic of an antiandrogenic mode of action, which is consistent with previously reported results in other species and supports the utility of the LAGDA design for chemical testing.

Differential transcript abundance in adipose tissue of mature beef cows during feed restriction and realimentation.

Feed costs account for over 70% of the annual expenditures in cow/calf production. During the production year the cow uses nutrients to support conceptus growth, milk production, work (grazing and locomotion), and maintenance requirements. The majority of the nutrients are used to support maintenance. Substrate cycling has been identified as one of the major contributors toward energy expenditure associated with maintenance in mature cows. The objective of this study was to determine whether beef cows that differ in the efficiency of weight gain differ in the relative abundance of transcripts for metabolic regulation in adipose tissue. Mature beef cows were subjected to feed restriction followed by ad libitum feed. Adipose tissue from twelve cows with high (n = 6) and low (n = 6) gain based on growth performance during the ad libitum feeding period was evaluated for transcriptome expression differences. A total of 496 genes were differentially expressed and passed Bonferroni correction for the animals with greater gain between restriction and realimentation and 491 genes were differentially expressed among animals with lesser gains between feed restriction and realimentation. Of these two differentially expressed gene lists, 144 genes were common between animals with greater and those with lesser gain. Enriched biological processes included the TCA cycle, oxidative phosphorylation, respiratory electron transport chain and fatty acid metabolic processes. Specific to adipose tissue of low gaining animals was glycolysis and to high gain animals was coenzyme, steroid, cellular amino acid, nitrogen compound metabolic processes, and sensory perception. The oxidative phosphorylation, mitochondrial dysfunction and cholesterol biosynthesis pathways were commonly associated with the high gain animals between feed restriction and realimentation, as well as with the low gaining animals between the two time points. Unique to the high gaining animals were valine degradation and LPS/IL-1 mediated inhibition of RXR function pathways. In this discovery study, genes involved in lipid metabolism, mitochondrial respiration and oxidative phosphorylation pathways appear to be critical to mature cows during times of abundant feed after feed restriction.

Metatranscriptomics profile of the gill microbial community during Bathymodiolus azoricus aquarium acclimatization at atmospheric pressure.

BackgroundThe deep-sea mussels Bathymodiolus azoricus (Bivalvia: Mytilidae) are the dominant macrofauna subsisting at the hydrothermal vents site Menez Gwen in the Mid-Atlantic Ridge (MAR). Their adaptive success in such challenging environments is largely due to their gill symbiotic association with chemosynthetic bacteria. We examined the response of vent mussels as they adapt to sea-level environmental conditions, through an assessment of the relative abundance of host-symbiont related RNA transcripts to better understand how the gill microbiome may drive host-symbiont interactions in vent mussels during hypothetical venting inactivity.ResultsThe metatranscriptome of B. azoricus was sequenced from gill tissues sampled at different time-points during a five-week acclimatization experiment, using Next-Generation-Sequencing. After Illumina sequencing, a total of 181,985,262 paired-end reads of 150 bp were generated with an average of 16,544,115 read per sample. Metatranscriptome analysis confirmed that experimental acclimatization in aquaria accounted for global gill transcript variation. Additionally, the analysis of 16S and 18S rRNA sequences data allowed for a comprehensive characterization of host-symbiont interactions, which included the gradual loss of gill endosymbionts and signaling pathways, associated with stress responses and energy metabolism, under experimental acclimatization. Dominant active transcripts were assigned to the following KEGG categories: “Ribosome”, “Oxidative phosphorylation” and “Chaperones and folding catalysts” suggesting specific metabolic responses to physiological adaptations in aquarium environment.ConclusionsGill metagenomics analyses highlighted microbial diversity shifts and a clear pattern of varying mRNA transcript abundancies and expression during acclimatization to aquarium conditions which indicate change in bacterial community activity. This approach holds potential for the discovery of new host-symbiont associations, evidencing new functional transcripts and a clearer picture of methane metabolism during loss of endosymbionts. Towards the end of acclimatization, we observed trends in three major functional subsystems, as evidenced by an increment of transcripts related to genetic information processes; the decrease of chaperone and folding catalysts and oxidative phosphorylation transcripts; but no change in transcripts of gluconeogenesis and co-factors-vitamins.

40 Gene Expression Profiling of In Vitro-Produced Blastocysts Derived from In Vitro-Matured Bovine Oocytes Vitrified/Warmed in Media Supplemented with a Biopolymer Produced by an Antarctic Bacterium

Previous reports have demonstrated the beneficial effect of antifreeze glycoprotein supplementation during oocyte vitrification on preventing ice crystal formation and thus enhancing developmental competence after vitrification-warming. Pseudomonas sp. ID1, a bacterium isolated from marine sediment from Antarctica, produces an exopolysaccharide, M1 EPS, as a cold adaptation mechanism. Despite numerous studies on structural and morphological damages induced by cryopreservation in oocytes, few studies have focused on the impact of vitrification on the expression pattern of genes during early embryo development. In the present study, the expression patterns of 6 genes (BAX, BCL2-like 1, DNMT3A, UBE2A, SCLC2A3, and HDAC1) were investigated in Day 8 blastocysts resulting from in vitro-matured oocytes vitrified/warmed in media supplemented with various concentrations of M1 EPS. After 21 h of IVM, 1,062 oocytes were vitrified/warmed in media supplemented with 0, 0.001, 0.01, 0.1, and 1 mg mL−1 M1 EPS. At 24 h of IVM, oocytes were in vitro fertilized and in vitro cultured and the resulting blastocysts were harvested at Day 8 for RNA extraction and qPCR analysis. Fresh, non-vitrified oocytes were used as a control. Analysis of gene expression was performed through Kruskall-Wallis test and followed by Mann-Whitney test, and the level of significance was set at P ≤ 0.05. No significant differences were detected in relative mRNA abundance for SLC2A3, UBE2A, or HDAC-1 between blastocysts derived from vitrified oocytes, regardless of M1 EPS treatment. Expression of DNMT3A was significantly higher in embryos obtained from oocytes vitrified and warmed with 0.1 mg mL−1 M1 EPS compared with other treatment groups. However, no differences in DNMT3A expression were observed when the other vitrified groups were compared. The relative abundance of BAX transcript in embryos from oocytes vitrified in media supplemented with 0.1 mg mL−1 M1 EPS was higher than that in 0 or 0.001 mg mL−1 groups. Embryos from 0.01 and 0.1 mg mL−1 groups showed higher BCL2-like 1 mRNA abundance than those from the 0, 0.001, and 1 mg mL−1 groups. Whereas blastocysts from oocytes vitrified with 0.01 mg mL−1 M1 EPS exhibited the lowest BAX:BCL2-like 1 ratio, no significant differences in BAX:BCL2-like 1 ratio were observed between the other treatments. The significantly lower BAX:BCL2 ratio observed in blastocysts obtained from oocytes vitrified with 0.01 mg mL−1 M1 EPS could be indicative for a better embryo quality. Although optimizing the use of M1 EPS may benefit oocyte cryopreservation protocols, further research is required to clarify the exact mechanism through which it exerts its protective role. This study was supported by the Spanish Ministry of Science and Innovation (Project AGL2016-79802-P and grant CTQ2014-59632-R).

173 Size Matters: a Follicle-of-Origin Effect on the Preovulatory Epigenetic Constitution of Germinal Vesicle-Stage Porcine Oocytes

The goal of this study was to evaluate global levels of a variety of histone modifications at different lysine (K) residues on Histone 3 (H3) within the chromatin of porcine germinal vesicle (GV)-stage oocytes that were aspirated from follicles of different sizes. We hypothesised that we would see evidence of a transition from open, transcriptionally active chromatin (in oocytes from smaller, growing follicles) to more closed, transcriptionally silent chromatin associated with fully grown oocytes (aspirated from large, preovulatory follicles). Cumulus-enclosed oocytes were aspirated from small (&lt;3 mm) or large (&gt;7 mm) follicles from abattoir-derived pig ovaries. Oocytes were denuded immediately after aspiration and then immunoprobed with antibodies specific for trimethylated (me3) H3K4, H3K9me3, and H3K27me3. Background-corrected nuclear fluorescence levels for each histone mark were collected from multiple oocytes from each of at least three experimental replicates (aspiration days). Data were subjected to one-way ANOVA with a Bonferroni multiple testing correction to determine whether there were differences in fluorescence intensities in the nuclei (germinal vesicles) of oocytes from small v. large follicles. Oocytes from large follicles displayed more intense nuclear staining for all 3 histone marks: average nuclear H3K4me3 intensity was 31.4% higher (P = 0.0004), H3K9me3 was 70.3% higher (P = 0.0218), and H3K27me3 was 32.0% higher (P = 0.0231) in oocytes from large follicles. An ancillary analysis of the data revealed no effect (P &gt; 0.1) of pubertal status (i.e. whether small and large follicles were aspirated from pre- v. post-pubertal ovaries) on the intensity of nuclear fluorescence for any of the marks evaluated. In continuation, 3 oocytes from both follicle types were collected on each of 6 aspiration days (i.e. 18 individual oocytes from each follicle type), and the mRNA from these were used for an RT-qPCR experiment to detect the relative abundance of transcripts from 21 different genes coding for histone methyltransferase or demethylase enzymes in oocytes from large v. small follicles. Of the 21 genes tested, 5 genes (KDM4C, KDM4D, KDM5B, KDM5C, and SETD7) were not detectable in our individual oocyte samples, but transcripts from 6 of the 16 remaining genes (KDM6A, KMT2B, MLL3, SETD1B, SETDB1, and SUV39H2) were shown to be significantly more abundant in oocytes from large follicles (at least 2-fold greater abundance and P &lt; 0.05). Although our expectation-that histone marks (and related transcripts) would consistently reflect a globally “repressive” chromatin configuration in oocytes from large follicles and a more “open” configuration in oocytes from small follicles-turned out to be untrue, the evidence suggests that the epigenetic constitution of oocytes from small follicles may indeed vary from that of oocytes from large, preovulatory follicles, and this phenomenon warrants further investigation.

Retention of the Structure and Function of Heparan Sulfate Biomaterials After Gamma Irradiation.

Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as "HS3") is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range of diseases and disorders.

Community structure of the metabolically active rumen bacterial and archaeal communities of dairy cows over the transition period.

Dairy cows experience dramatic changes in host physiology from gestation to lactation period and dietary switch from high-forage prepartum diet to high-concentrate postpartum diet over the transition period (parturition +/- three weeks). Understanding the community structure and activity of the rumen microbiota and its associative patterns over the transition period may provide insight for e.g. improving animal health and production. In the present study, rumen samples from ten primiparous Holstein dairy cows were collected over seven weeks spanning the transition period. Total RNA was extracted from the rumen samples and cDNA thereof was subsequently used for characterizing the metabolically active bacterial (16S rRNA transcript amplicon sequencing) and archaeal (qPCR, T-RFLP and mcrA and 16S rRNA transcript amplicon sequencing) communities. The metabolically active bacterial community was dominated by three phyla, showing significant changes in relative abundance range over the transition period: Firmicutes (from prepartum 57% to postpartum 35%), Bacteroidetes (from prepartum 22% to postpartum 18%) and Proteobacteria (from prepartum 7% to postpartum 32%). For the archaea, qPCR analysis of 16S rRNA transcript number, revealed a significant prepartum to postpartum increase in Methanobacteriales, in accordance with an observed increase (from prepartum 80% to postpartum 89%) in relative abundance of 16S rRNA transcript amplicons allocated to this order. On the other hand, a significant prepartum to postpartum decrease (from 15% to 2%) was observed in relative abundance of Methanomassiliicoccales 16S rRNA transcripts. In contrast to qPCR analysis of the 16S rRNA transcripts, quantification of mcrA transcripts revealed no change in total abundance of metabolically active methanogens over the transition period. According to T-RFLP analysis of the mcrA transcripts, two Methanobacteriales genera, Methanobrevibacter and Methanosphaera (represented by the T-RFs 39 and 267 bp), represented more than 70% of the metabolically active methanogens, showing no significant changes over the transition period; minor T-RFs, likely to represent members of the order Methanomassiliicoccales and with a relative abundance below 5% in total, decreased significantly over the transition period. In accordance with the T-RFLP analysis, the mcrA transcript amplicon sequencing revealed Methanobacteriales to cover 99% of the total reads, dominated by the genera Methanobrevibacter (75%) and Methanosphaera (24%), whereas the Methanomassiliicoccales order covered only 0.2% of the total reads. In conclusion, the present study showed that the structure of the metabolically active bacterial and archaeal rumen communities changed over the transition period, likely in response to the dramatic changes in physiology and nutritional factors like dry matter intake and feed composition. It should be noted however that for the methanogens, the observed community changes were influenced by the analyzed gene (mcrA or 16S rRNA).

Evidence of high transport and phosphorylation capacity for both glucose and fructose in the ruby-throated hummingbird (Archilochus colubris).

Hummingbirds are able to fuel hovering flight entirely with recently ingested glucose or fructose. Among vertebrates, several steps of sugar flux from circulation to skeletal muscle are potentially rate-limiting, including transport into muscle and subsequent phosphorylation. While capacities for glucose flux are substantial, capacities for fructose flux are comparatively low. The mechanisms underlying apparent high rates of glucose and fructose oxidation in hummingbird flight muscle remain unclear. We examined relative expression of facilitative sugar transporters (GLUTs) and enzymes of fructolysis in ruby-throated hummingbird (Archilochus colubris) tissues involved in energy homeostasis and flight, via qPCR and measured hexokinase activity in pectoralis in vitro. We hypothesized that expression of these genes was upregulated in hummingbird flight muscle compared to other vertebrates. We found that hummingbird pectoralis had high relative transcript abundance of GLUT1 and GLUT5 compared to expression profiles of other vertebrates. In particular, GLUT5 expression in pectoralis was similar to that of intestine. We demonstrated minimal relative densities of fructolytic enzymes in pectoralis, suggesting that the ketohexokinase pathway does not rapidly metabolize fructose in these muscles. Instead, we found that the capacity for phosphorylation of either glucose or fructose by hexokinase is very high in pectoralis in vitro. The contributions of individual hexokinase isoforms remain to be determined. Our results further characterize the strategies by which hummingbirds, and perhaps other nectarivores, accomplish rapid sugar flux. High transport and sugar phosphorylation capacities appear to exist in flight muscle, though the enzymatic pathways that catalyze the phosphorylation of sugar in muscle remain uncertain.

The CXCL12-CXCR4 signaling promotes oocyte maturation by regulating cumulus expansion in sheep

Gonadotropins and growth factors synergistically regulate folliculogenesis and oocyte development. C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4 are expressed in ovaries of sheep, cattle and other species, however, roles of this multifunctional signal axis in oocyte maturation are not defined. Using sheep as a model, we examined the expression patterns and functions of the CXCL12-CXCR4 axis during oocyte maturation. CXCL12 and CXCR4 mRNA and protein were present in oocytes and granulosa cells. Relative abundance of CXCR4 transcript was controlled by epidermal growth factor (EGF). Transient inhibition of CXCR4 suppressed oocyte nuclear maturation while supplementing recombination CXCL12 significantly increased percent of oocyte undergone metaphase I phase. Inhibition of CXCR4 function decreased cumulus expansion growth rate. Furthermore, granulosa cell migration was impaired and expression of hyaluronan synthase 2 (HAS2) and hyaluronan binding protein tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) were downregulated by CXCR4 inhibition. These findings revealed a novel role of the CXCL12-CXCR4 signaling in oocyte development in sheep.

Metabolic and molecular responses to calcium soap of fish oil fed to ewes during peripartal period.

It has been shown that n-3 long chain fatty acids (n-3 LCFA) are involved in energy/lipid metabolisms, reproductive parameters, and molecular regulations leading to maintained homeostasis. We hypothesized that supplementation of peripartal diets with fish oil (FO), as a source of n-3 LCFA, could improve energy balance and modulate metabolic pressure in a sheep model. Prepartum ewes (n = 24) were fed control (CON) or calcium soap of fish oil (FO) supplemented-diet from four weeks before until three weeks after parturation. Feed intake, body weight (BW) change, plasma metabolites, colostrums/milk composition, and fatty acids profile of milk along with the expression of core microRNAs in glucose and lipid metabolism were evaluated. Prepartal feed intake decreased in FO group (1674 ± 33.26 vs. 1812 ± 35.56) though post-partal intake was similar. Differences in BW were not also significant (55.47 ± 2.07 in CON vs. 53.69 ± 1.94 in FO). No differences were observed in plasma metabolites except for cholesterol that was lower in FO group (56.25 ± 0.71 vs. 53.09 ± 0.61). Milk fat percentage was reduced (8.82 ± 0.49 vs. 7.03 ± 0.45) while the percentage of milk total n-3 LCFA increased in FO group. In accordance, the relative transcript abundance of miR-101 (0.215 ± 0.08) and miR-103 (0.37 ± 0.15) decreased by FO supplementation. Results showed that FO supplementation during peripartal period decreased milk fat, feed intake, plasma cholesterol, milk n-6:n-3 ratio and the expression of miR-101. Although the trend indicated that FO could alter lipid metabolism during transition period, further studies are needed to fully address its effect on energy balance and homeorhetic processes.

Deciphering the olfactory repertoire of the tiger mosquito Aedes albopictus

BackgroundThe Asian tiger mosquito Aedes albopictus is a highly invasive species and competent vector of several arboviruses (e.g. dengue, chikungunya, Zika) and parasites (e.g. dirofilaria) of public health importance. Compared to other mosquito species, Ae. albopictus females exhibit a generalist host seeking as well as a very aggressive biting behaviour that are responsible for its high degree of nuisance. Several complex mosquito behaviours such as host seeking, feeding, mating or oviposition rely on olfactory stimuli that target a range of sensory neurons localized mainly on specialized head appendages such as antennae, maxillary palps and the mouthparts.ResultsWith the aim to describe the Ae. albopictus olfactory repertoire we have used RNA-seq to reveal the transcriptome profiles of female antennae and maxillary palps. Male heads and whole female bodies were employed as reference for differential expression analysis. The relative transcript abundance within each tissue (TPM, transcripts per kilobase per million) and the pairwise differential abundance in the different tissues (fold change values and false discovery rates) were evaluated. Contigs upregulated in the antennae (620) and maxillary palps (268) were identified and relative GO and PFAM enrichment profiles analysed. Chemosensory genes were described: overall, 77 odorant binding proteins (OBP), 82 odorant receptors (OR), 60 ionotropic receptors (IR) and 30 gustatory receptors (GR) were identified by comparative genomics and transcriptomics. In addition, orthologs of genes expressed in the female/male maxillary palps and/or antennae and involved in thermosensation (e.g. pyrexia and arrestin1), mechanosensation (e.g. piezo and painless) and neuromodulation were classified.ConclusionsWe provide here the first detailed transcriptome of the main Ae. albopictus sensory appendages, i.e. antennae and maxillary palps. A deeper knowledge of the olfactory repertoire of the tiger mosquito will help to better understand its biology and may pave the way to design new attractants/repellents.

Shifts in the community composition of methane-cycling microorganisms during lake shrinkage

Intensification of anthropogenic activities has resulted in shrinkage of lakes, which are important sources and sinks of the greenhouse gas methane. Given the increasing interest in the potential importance of CH4 flux, we employed Illumina Miseq pyrosequencing to examine the effect of lake shrinkage (in a lake that had decreased from 3.7km2 to 3.0km2 in area during the last 20years) on the community abundance, activity and structure of methane-cycling microorganisms under four types of soils during lake recession: Sediment (the sediment of the lake, at the maximum depth of 6m), Slope (soil at a lake water depth of 0.6m), Marsh (the seasonally flooded marsh and wetland formed after recession of the lake) and Meadow (the meadow formed after recession of the lake, about 200m from the lake). The results showed that lake shrinkage decreased the relative abundance and transcripts of soil methanogens and methanotrophs along the gradient from Sediment to Slope to Marsh. Meadow, having the longest drying time, had both higher soil nutrient concentrations (including dissolved organic carbon, total carbon and total nitrogen), and higher relative abundance of 16S rRNA genes and transcripts of methanogens and methanotrophs. Results of methanogen community structure analysis indicated ‘resistance’ to change under lake shrinkage, with all communities dominated by Methanosaetaceae except the Slope community, which was dominated by Methanoregulaceae, while the dominant methanotroph changed from Methylomicrobium (a Type I methanotroph) (in Sediment, Slope and Marsh) to Methylocaldum (Type I) and Methylosinus (Type II) (in Meadow) under lake shrinkage. Taken together, the microbial community data indicate that CH4 production and consumption potentially recedes under lake shrinkage, whereas the formation of Meadow may increase CH4 emission and oxidization potential.

Transcriptome-Based Identification of the Optimal Reference CHO Genes for Normalisation of qPCR Data.

Real-time quantitative PCR (qPCR) is the standard method for determination of relative changes in mRNA transcript abundance. Analytical accuracy, precision and reliability are critically dependent on the selection of internal control reference genes. In this study, the authors have identified optimal reference genes that can be utilised universally for qPCR analysis of CHO cell mRNAs. Initially, transcriptomic datasets were analysed to identify eight endogenous genes that exhibited high expression stability across four distinct CHO cell lines sampled in different culture phases. The relative transcript abundance of each gene in 20 diverse, commonly applied experimental conditions was then determined by qPCR analysis. Utilizing GeNorm, BestKeeper and NormFinder algorithms, the authors identified four mRNAs (Gnb1, Fkbp1a, Tmed2 and Mmadhc) that exhibited a highly stable level of expression across all conditions, validating their utility as universally applicable reference genes. Whilst any combination of only two genes can be generally used for normalisation of qPCR data, the authors show that specific combinations of reference genes are particularly suited to discrete experimental conditions. In summary, the authors report the identification of fully validated universal reference genes, optimised primer sequences robust to genomic mutations and simple reference gene pair selection guidelines that enable streamlined qPCR analyses of mRNA abundance in CHO cells with maximum accuracy and precision.

Total rRNA-Seq Analysis Gives Insight into Bacterial, Fungal, Protozoal and Archaeal Communities in the Rumen Using an Optimized RNA Isolation Method.

Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, 44.16 ± 1.55, 35.38 ± 1.64, and 16.37 ± 0.65% respectively. This represents the first study to define the relative active contributions of these populations to the rumen ecosystem and is especially important in defining the role of the anaerobic fungi and protozoa.

Osmium‐Mediated Transformation of 4‐Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing

AbstractTo understand the functional roles of RNA in the cell, it is essential to elucidate the dynamics of their production, processing and decay. A recent method for assessing mRNA dynamics is metabolic labeling with 4‐thiouridine (4sU), followed by thio‐selective attachment of affinity tags. Detection of labeled transcripts by affinity purification and hybridization to microarrays or by deep sequencing then reveals RNA expression levels. Here, we present a novel sequencing method (TUC‐seq) that eliminates affinity purification and allows for direct assessment of 4sU‐labeled RNA. It employs an OsO4‐mediated transformation to convert 4sU into cytosine. We exemplify the utility of the new method for verification of endogenous 4sU in tRNAs and for the detection of pulse‐labeled mRNA of seven selected genes in mammalian cells to determine the relative abundance of the new transcripts. The results prove TUC‐seq as a straight‐forward and highly versatile method for studies of cellular RNA dynamics.

Osmium-Mediated Transformation of 4-Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing.

To understand the functional roles of RNA in the cell, it is essential to elucidate the dynamics of their production, processing and decay. A recent method for assessing mRNA dynamics is metabolic labeling with 4-thiouridine (4sU), followed by thio-selective attachment of affinity tags. Detection of labeled transcripts by affinity purification and hybridization to microarrays or by deep sequencing then reveals RNA expression levels. Here, we present a novel sequencing method (TUC-seq) that eliminates affinity purification and allows for direct assessment of 4sU-labeled RNA. It employs an OsO4 -mediated transformation to convert 4sU into cytosine. We exemplify the utility of the new method for verification of endogenous 4sU in tRNAs and for the detection of pulse-labeled mRNA of seven selected genes in mammalian cells to determine the relative abundance of the new transcripts. The results prove TUC-seq as a straight-forward and highly versatile method for studies of cellular RNA dynamics.

Identification of optimal reference genes for examination of gene expression in different tissues of fetal yaks

Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used to study the relative abundance of mRNA transcripts because of its sensitivity and reliable quantification. However, the reliability of the interpretation of expression data is influenced by several complex factors, including RNA quality, transcription activity, and PCR efficiency, among others. To avoid experimental errors arising from potential variation, the selection of appropriate reference genes to normalize gene expression is essential. In this study, 10 commonly used reference genes – ACTB, B2M, HPRT1, GAPDH, 18SrRNA, 28SrRNA, PPIA, UBE2D2, SDHA, and TBP – were selected as candidate reference genes for six fetal tissues (heart, liver, spleen, lung, kidney, and forehead skin) of yak (Bos grunniens). The transcription stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper. The results showed that the combination of TBP and ACTB provided high-quality data for further study. In contrast, the commonly used reference genes 28SrRNA, SDHA, GAPDH, and B2M should not be used for endogenous controls because of their unstable expression in this study. The reference genes that could be used in future gene expression studies in yaks were indentified.

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress

BackgroundHigh ambient temperature is known to affect fish gonadal development and physiology in a variety of ways depending on the severity and duration of exposure; however, the underlying molecular mechanisms are poorly understood. Gonadal gene expression influence the gonadal development, physiology and the quality of egg/sperm produced in teleosts and the mechanistic understanding of spatio-temporal changes in the gonadal gene expression could be instrumental in controlling the fate of egg/sperm and the quality of seed produced. Real time-quantititative polymerase chain reaction (RT-qCR), is a high throughput, sensitive and reproducible methodology used for understanding gene expression patterns by measuring the relative abundance of mRNA transcripts. However, its accuracy relies upon a suitable reference gene whose expression levels remain stable across various experimental conditions. In the present study, we evaluated the suitability of ten potential reference genes to be used as internal controls in RT-qPCR analysis in gonadal tissues (ovary and testis) of minnow Puntius sophore exposed to high temperature stress for different time periods (7 days, 60 days). Expression analysis of ten different constitutively expressed genes viz. 18S ribosomal RNA (18S rRNA), beta actin (βactin), β-2 microglobulin (b2mg), eukaryotic elongation factor-1 (eef1), glyceraldehyde-3phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), ribosomal binding protein L13 (rpl13), tubulin (tub), tata box binding protein (tbp), ubiquitin (ubi) was carried out by using RT-qPCR and the stability in their expressions were evaluated by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder.ResultsIn ovary, eef1 was found to be the most suitable reference gene in all the algorithms used. In testis, b2mg was found to be the most suitable reference gene in delta Ct, BestKeeper, NormFinder analysis while tbp and eef1 were found to be the most suitable reference genes in geNorm analysis.ConclusionsIn conclusion, eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively, of Puntius sophore exposed to high temperature stress, and could be used as internal controls for gene expression analysis in gonadal tissues of Puntius sophore.

Dietary Supplementation of Algae-Derived Omega-3 Fatty Acids Influences Endometrial and Conceptus Transcript Profiles in Mares

Maternal diet has been shown to impact fetal development across a variety of species. Given the reported effects of omega-3 long-chain polyunsaturated fatty acid (n-3 LCPUFA) supplementation on health across species, it was hypothesized that supplementation of algal-based n-3 LCPUFA to mares around conception would alter the maternal uterine environment and modify gene expression in early conceptuses. Nonlactating, light horse mares were supplemented with concentrate (n = 6) or an isocaloric diet containing 0.06 g/kg body weight n-3 LCPUFA (primarily docosahexaenoic acid [DHA], n = 7) at least 60 days before first sample collection. Four consecutive ovulatory cycles were monitored. Uterine endometrial samples were obtained 12.5 days postovulation from cycles 1 (uninseminated), 3, and 4 (bred to a common stallion); embryos were flushed from cycles 3 and 4. Biopsies from supplemented mares were greater in DHA and total omega-3 fatty acid concentrations (P < .05), indicating successful tissue incorporation. Uninseminated n-3 LCPUFA supplemented mares displayed increased PTGFS and reduced IL6 gene expression (P < .01) than controls. Supplemented pregnant mares had increased mRNA abundance of PTGES (P = .05), PTGFS (P = .03), and SLCO2A1 (P = .01), while supplemented barren mares exhibited lower PTGFS (P = .05) and IL6 (P < .01) expression than control mares. In the conceptus, relative transcript abundance of trophoblast and endoderm markers (GATA3, GATA4, GATA6, TFAP2A, ELF3) was greater (P < .05) in embryos from supplemented mares. These results indicate algae-derived n-3 LCPUFA supplementation prior to breeding alters the maternal uterine environment and modifies expression of genes in preimplantation equine conceptuses.