Relative Transcript Abundance Research Articles

253 EFFECTS OF ADDITION OF ALPHA-LINOLENIC ACID INTO MATURATION MEDIUM ON IN VITRO DEVELOPMENT AND EXPRESSION OF APOPTOSIS-RELATED GENES OF GOAT EMBRYOS

Fatty acids are important sources of energy for oocytes and embryos. In bovine, an increased level of rumen-inert fatty acids in the diet improved the developmental competence of oocytes to the blastocyst stage and also embryo quality. As described in our previous report, providing appropriate levels of α-linolenic acid (ALA) in maturation medium had beneficial effects on nuclear maturation and embryonic development in the goat. Considering these beneficial effects, here we have conducted experiments to evaluate whether addition of ALA to goat oocyte maturation medium can affect the quality of blastocyst and the transcription of apoptosis-related Bax, Bcl-2, and p53 genes. Ovaries were collected from goats, and cumulus-oocyte complexes (COC) were recovered by the slicing method. The COC were placed in maturation medium supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, oocytes from both the treatment (n = 113) and control (n = 104) groups were subjected to IVF followed by culture in CR1aa medium for 8 days under the conditions stated above. The cleavage and blastocyst rates were recorded at Days 3 and 8 of culture, respectively. To examine the effect of ALA on total cell number and apoptosis of the blastocyst cells, the blastocysts from 50 μM ALA-treated and control oocytes were stained with 4′,6-diamidino-2-phenylindole to count total cell number, and apoptotic cells in these blastocysts were detected with TUNEL assay. Blastocysts derived either from 50 μM ALA-treated oocytes or control oocytes were also evaluated for the expression of Bax, Bcl-2, and p53 genes. The cleavage and blastocyst rates were compared by chi-square analysis. Differentially expressed genes were analysed by 1-way ANOVA. A P-value of less than 0.05 was considered significant. Although cleavage rates after IVF were similar (P > 0.05) between 50 μM ALA-treated (68.1%) and control (55.8%) groups, 50 μM ALA-treated oocytes produced more (25.7%) blastocysts than the control group (13.5%; P < 0.05). Blastocysts derived from oocytes supplemented with 50 μM ALA not only had a greater (P < 0.05) total cell number (115.2), but also a lower (P < 0.05) number of apoptotic cells (3.1) compared with the control blastocysts (110.8 and 4.2, respectively). The relative transcript abundance of Bax and p53 was decreased (P < 0.05) in blastocysts that originated from the 50 μM ALA group compared with control blastocysts. Furthermore, there was an increased (P < 0.05) expression of Bcl-2 transcripts in blastocysts derived from the 50 μM ALA-treated oocytes compared with the control. In conclusion, our findings revealed that ALA-treated medium led to an improvement in blastocyst rate and quality as determined by greater total cell number, lower number of apoptotic cells, and altered expression of apoptosis-related genes.

Molecular Landscape of Helopeltis theivora Induced Transcriptome and Defense Gene Expression in Tea

Tea is the second most consumed beverage worldwide whose cultivation is greatly challenged by a large variety of biotic and abiotic stresses. Among the biotic factors, the hemipteran pest Helopeltis theivora Waterhouse is particularly devastating in Asia and Africa, rendering the crop unsuitable for any downstream processing. The present study, for the first time, endeavors to dissect the molecular events associated with such infestations and identify potential molecular protagonists influencing Helopeltis tolerance in Assam tea. We analyzed the transcriptome of infested tea plants having contrasting responses to pest feeding by suppression subtractive hybridization and quantified the relative abundances of defense-associated transcripts. We analyzed 445 unigenes derived from 1558 expressed sequence tags (ESTs) from three different infestation conditions. Our study indicate that defense responses to the pest greatly vary in temporal and cultivar-specific manner characterized by an exclusive upregulation of specific defense-associated genes in the tolerant cultivar. Moreover, it was observed that transcripts related to flavonoid biosynthesis, purine metabolism, formate metabolism, jasmonic acid biosynthesis and signaling, and cell wall metabolism are uniquely enriched in the same. Interestingly, those involved in caffeine biosynthesis remain comparatively low and unaltered. We report higher trichome densities on the abaxial surface of the leaf in tolerant cultivar while puncture causes their tender reorientation. SEM of the insect mouthparts reveal higher concentrations of sensilla-like structures near the distal end and tip of the proboscis which are known to act as chemoreceptors in other hemipterans. Our investigation provides the first ever glimpse of Helopeltis-induced defense gene network and provides the much needed platform for improvement of the crop for better Helopeltis tolerance through breeding and transgenic approaches. On a wider perspective, the study will augment our understanding of molecular responses to phloem herbivory in plants.

Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART).

Leptin Gene Expression in Rabbits during Pregnancy And Fetal Life

Leptin may act as the critical link between adipose tissue and reproduction.Although considerable progress has been achieved in understanding the reproductive actions of leptin, much work is needed for understanding its physiological role. Till now, no data has been published about the distribution and expression levels of leptin in the rabbit maternal adipose tissue, placenta, and various fetal rabbit tissues during pregnancy and postpartum. Our results indicated that circulating leptin levels in rabbit serum during pregnancy were significantly higher than that in postpartum and non pregnant rabbits. Furthermore, leptin showed positive correlations with body weight in pregnant and postpartum rabbits. RT-PCR verified the presence of RNAs encoding leptin in the rabbit maternal perirenal adipose tissue, placenta, and several fetal tissues; including brain, liver, adipose tissue, and bone. The relative abundance of leptin RNA in rabbit maternal adipose tissue was significantly higher at 20th day of pregnancy than that of non pregnant rabbits, while it was significantly decreased at 2nd day after parturition. No significant changes in the placental leptin RNA levels were noticed in pregnant rabbits at 10th, 20th, and 30th day of pregnancy. The relative abundance of fetal leptin transcripts at day 30th of pregnancy was in the order of liver> bone ≥ adipose tissue > brain. The present study provides new evidence for the distribution and expression levels of leptin in the rabbit maternal and fetal tissues during pregnancy and supports the importance of leptin in reproductive physiology and fetal development.

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways.

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation.

Evidence of novel phylogenetic lineages of methanogenic archaea from hypersaline microbial mats.

Methanogenesis in hypersaline and high-sulfate environments is typically dominated by methylotrophic methanogens because sulfate reduction is thermodynamically favored over hydrogenotrophic methanogenesis in these environments. We characterized the community composition of methanogenic archaea in both unmanipulated and incubated microbial mats from different hypersaline environments in Baja California Sur, Mexico. Clone libraries of methyl coenzyme-M reductase (mcrA) sequences and DGGE band patterns of 16S rRNA and mcrA sequences showed that the methanogen community in these microbial mats is dominated by methylotrophic methanogens of the genus Methanohalophilus. However, phylogenetic analyses of mcrA sequences from these mats also revealed two new lineages corresponding to putative hydrogenotrophic methanogens related with the strictly hydrogenotrophic order Methanomicrobiales. Stimulated methane production under decreased salinity and sulfate concentrations also suggested the presence of hydrogenotrophic methanogens in these samples. The relative abundance of mcrA gene and transcripts, estimated by SYBR green I qPCR assays, suggested the activity of different phylogenetic groups of methanogens, including the two novel clusters, in unmanipulated samples of hypersaline microbial mats. Using geochemical and molecular approaches, we show that substrate limitation and values of salinity and sulfate higher than 3 % and 25 mM (respectively) are potential environmental constraints for methanogenesis in these environments. Microcosm experiments with modifications of salinity and sulfate concentrations and TMA addition showed that upper salt and sulfate concentrations for occurrence of methylotrophic methanogenesis were 28 % and 263 mM, respectively. This study provides phylogenetic information about uncultivated and undescribed methanogenic archaea from hypersaline environments.

Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes

Hot season is a major constraint to production and reproduction in buffaloes. The present work aimed to investigate the effect of season on ovarian function, developmental competence, and the relative abundance of gene expression in buffalo oocytes. Three experiments were conducted. In experiment 1, pairs of buffalo ovaries were collected during cold season (CS, autumn and winter) and hot season (HS, spring and summer), and the number of antral follicles was recorded. Cumulus oocyte complexes (COCs) were aspirated and evaluated according to their morphology into four Grades. In experiment 2, Grade A and B COCs collected during CS and HS were in vitro matured (IVM) for 24 hours under standard conditions at 38.5 °C in a humidified air of 5% CO2. After IVM, cumulus cells were removed and oocytes were fixed, stained with 1% aceto-orcein, and evaluated for nuclear configuration. In vitro matured buffalo oocytes harvested during CS or HS were in vitro fertilized (IVF) using frozen-thawed buffalo semen and cultured in vitro to the blastocyst stage. In experiment 3, buffalo COCs and in vitro matured oocytes were collected during CS and HS, and then snap frozen in liquid nitrogen for gene expression analysis. Total RNA was extracted from COCs and in vitro matured oocytes, and complementary DNA was synthesized; quantitative Reverse Transcription-Polymerase Chain Reaction was performed for eight candidate genes including GAPDH, ACTB, B2M, GDF9, BMP15, HSP70, and SOD2. The results indicated that HS significantly (P < 0.01) decreased the number of antral follicles and the number of COCs recovered per ovary. The number of Grade A, B, and C COCs was lower (P < 0.05) during HS than CS. In vitro maturation of buffalo oocytes during HS significantly (P < 0.01) reduced the number of oocytes reaching the metaphase II stage and increased the percentage of degenerated oocytes compared with CS. Oocytes collected during HS also showed signs of cytoplasmic degeneration. After IVF, cleavage rate was lower (P < 0.01) for oocytes collected during HS, and the percentage of oocytes arrested at the two-cell stage was higher (P < 0.01) than oocytes IVF during CS. Oocytes matured during CS showed a higher (P < 0.01) blastocyst rate than those matured during HS. Also, COCs recovered in HS showed significant (P < 0.05) upregulation of HSP70 mRNA expression compared with those recovered in CS. For in vitro matured oocytes, CS down regulated the transcript abundance of ACTB and upregulated GAPDH and HSP70 mRNA levels compared with HS condition. In conclusion, HS could impair buffalo fertility by reducing the number of antral follicles and oocyte quality. In vitro maturation of buffalo oocytes during HS impairs their nuclear and cytoplasmic maturation, fertilization, and subsequent embryo development to the morula and blastocyst stages. This could be in part because of the altered gene expression found in COCs and in vitro matured oocytes.

Metatranscriptomic analysis of arctic peat soil microbiota.

Recent advances in meta-omics and particularly metatranscriptomic approaches have enabled detailed studies of the structure and function of microbial communities in many ecosystems. Molecular analyses of peat soils, ecosystems important to the global carbon balance, are still challenging due to the presence of coextracted substances that inhibit enzymes used in downstream applications. We sampled layers at different depths from two high-Arctic peat soils in Svalbard for metatranscriptome preparation. Here we show that enzyme inhibition in the preparation of metatranscriptomic libraries can be circumvented by linear amplification of diluted template RNA. A comparative analysis of mRNA-enriched and nonenriched metatranscriptomes showed that mRNA enrichment resulted in a 2-fold increase in the relative abundance of mRNA but biased the relative distribution of mRNA. The relative abundance of transcripts for cellulose degradation decreased with depth, while the transcripts for hemicellulose debranching increased, indicating that the polysaccharide composition of the peat was different in the deeper and older layers. Taxonomic annotation revealed that Actinobacteria and Bacteroidetes were the dominating polysaccharide decomposers. The relative abundances of 16S rRNA and mRNA transcripts of methanogenic Archaea increased substantially with depth. Acetoclastic methanogenesis was the dominating pathway, followed by methanogenesis from formate. The relative abundances of 16S rRNA and mRNA assigned to the methanotrophic Methylococcaceae, primarily Methylobacter, increased with depth. In conclusion, linear amplification of total RNA and deep sequencing constituted the preferred method for metatranscriptomic preparation to enable high-resolution functional and taxonomic analyses of the active microbiota in Arctic peat soil.

Sequence and expression analyses of ethylene response factors highly expressed in latex cells from Hevea brasiliensis.

The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors.

Overexpression of AtSTO1 leads to improved salt tolerance in Populus tremula×P. alba.

One of the major abiotic stress conditions limiting healthy growth of trees is salinity stress. The use of gene manipulation for increased tolerance to abiotic stress has been successful in many plant species. Overexpression of the Arabidopsis SALT TOLERANT1 (STO1) gene leads to increased concentrations of 9-cis-epoxycarotenoid dioxygenase3, a vital enzyme in Arabidopsis abscisic acid biosynthesis. In the present work, the Arabidopsis STO1 gene (AtSTO1) was overexpressed in poplar to determine if the transgene would confer enhanced salt tolerance to the generated transgenics. The results of multiple greenhouse trials indicated that the transgenic poplar lines had greater levels of resistance to NaCl than wild-type plants. Analysis using RT-PCR indicated a variation in the relative abundance of the STO1 transcript in the transgenics that coincided with tolerance to salt. Several physiological and morphological changes such as greater overall biomass, greater root biomass, improved photosynthesis, and greater pith size were observed in the transgenics when compared to controls undergoing salt stress. These results indicated overexpression of AtSTO1 improved salt tolerance in poplar.

Adipose and muscle tissue profile of CD36 transcripts in obese subjects highlights the role of CD36 in fatty acid homeostasis and insulin resistance.

OBJECTIVEFatty acid (FA) metabolism is tightly regulated across several tissues and impacts insulin sensitivity. CD36 facilitates cellular FA uptake, and CD36 genetic variants associate with lipid abnormalities and susceptibility to metabolic syndrome. The objective of this study was to gain insight regarding the in vivo metabolic influence of muscle and adipose tissue CD36. For this, we determined the relationships between CD36 alternative transcripts, which can reflect tissue-specific CD36 regulation, and measures of FA metabolism and insulin resistance.RESEARCH DESIGN AND METHODSThe relative abundance of alternative CD36 transcripts in adipose tissue and skeletal muscle from 53 nondiabetic obese subjects was measured and related to insulin sensitivity and FA metabolism assessed by hyperinsulinemic–euglycemic clamps and isotopic tracers for glucose and FA.RESULTSTranscript 1C, one of two major transcripts in adipose tissue, that is restricted to adipocytes predicted systemic and tissue (adipose, liver, and muscle) insulin sensitivity, suggesting adipocyte CD36 protects against insulin resistance. Transcripts 1B and 1A, the major transcripts in skeletal muscle, correlated with FA disposal rate and triglyceride clearance, supporting importance of muscle CD36 in clearance of circulating FA. Additionally, the common CD36 single nucleotide polymorphism rs1761667 selectively influenced CD36 transcripts and exacerbated insulin resistance of glucose disposal by muscle.CONCLUSIONSAlternative CD36 transcripts differentially influence tissue CD36 and consequently FA homeostasis and insulin sensitivity. Adipocyte CD36 appears to be metabolically protective, and its selective upregulation might have therapeutic potential in insulin resistance.

Metatranscriptome profiling of a harmful algal bloom

Metagenomic methods provide a powerful means to investigate complex ecological phenomena. Developed originally for study of Bacteria and Archaea, the application of these methods to eukaryotic microorganisms is yet to be fully realized. Most prior environmental molecular studies of eukaryotes have relied heavily on PCR amplification with eukaryote-specific primers. Here we apply high throughput short-read sequencing of poly-A selected RNA to capture the metatranscriptome of an estuarine dinoflagellate bloom. To validate the metatranscriptome assembly process we simulated metatranscriptomic datasets using short-read sequencing data from clonal cultures of four algae of varying phylogenetic distance. We find that the proportion of chimeric transcripts reconstructed from community transcriptome sequencing is low, suggesting that metatranscriptomic sequencing can be used to accurately reconstruct the transcripts expressed by bloom-forming communities of eukaryotes. To further validate the bloom metatransciptome assembly we compared it to a transcriptomic assembly from a cultured, clonal isolate of the dominant bloom-causing alga and found that the two assemblies are highly similar. Eukaryote-wide phylogenetic analyses reveal the taxonomic composition of the bloom community, which is comprised of several dinoflagellates, ciliates, animals, and fungi. The assembled metatranscriptome reveals the functional genomic composition of a metabolically active community. Highlighting the potential power of these methods, we found that relative transcript abundance patterns suggest that the dominant dinoflagellate might be expressing toxin biosynthesis related genes at a higher level in the presence of competitors, predators and prey compared to it growing in monoculture.

Analysis of chemosensory gene families in the beetle Monochamus alternatus and its parasitoid Dastarcus helophoroides

We assembled antennal transcriptomes of pest Monochamus alternatus and its parasitoid Dastarcus helophoroides to identify the members of the major chemosensory multi-gene families. Gene ontology (GO) annotation indicated that the relative abundance of transcripts associated with specific GO terms was highly similar in the two species. In chemosensory gene families, we identified 52 transcripts encoding putative odorant-binding proteins (OBPs), 19 chemosensory proteins (CSPs), 10 olfactory receptors (ORs), 8 ionotropic receptors (IRs), 2 gustatory receptors (GRs), and 5 sensory neuron membrane proteins (SNMPs) in these two transcriptomes. Predicted protein sequences were compared with Dendroctonus ponderosae, Tribolium castaneum and Drosophila melanogaster. The results of phylogenetic trees showed that some clusters included only OBPs or CSPs from D. helophoroides, some clusters included only OBPs or CSPs from M. alternatus, while some clusters included OBPs or CSPs from both M. alternatus and D. helophoroides. The identification of the chemosensory genes and the phylogenetic relationship of these genes between two species might provide new ideas for controlling M. alternatus and improving current strategies for biological control.

Response of a rice paddy soil methanogen to syntrophic growth as revealed by transcriptional analyses.

Members of Methanocellales are widespread in paddy field soils and play the key role in methane production. These methanogens feature largely in these organisms’ adaptation to low H2 and syntrophic growth with anaerobic fatty acid oxidizers. The adaptive mechanisms, however, remain unknown. In the present study, we determined the transcripts of 21 genes involved in the key steps of methanogenesis and acetate assimilation of Methanocella conradii HZ254, a strain recently isolated from paddy field soil. M. conradii was grown in monoculture and syntrophically with Pelotomaculum thermopropionicum (a propionate syntroph) or Syntrophothermus lipocalidus (a butyrate syntroph). Comparison of the relative transcript abundances showed that three hydrogenase-encoding genes and all methanogenesis-related genes tested were upregulated in cocultures relative to monoculture. The genes encoding formylmethanofuran dehydrogenase (Fwd), heterodisulfide reductase (Hdr), and the membrane-bound energy-converting hydrogenase (Ech) were the most upregulated among the evaluated genes. The expression of the formate dehydrogenase (Fdh)-encoding gene also was significantly upregulated. In contrast, an acetate assimilation gene was downregulated in cocultures. The genes coding for Fwd, Hdr, and the D subunit of F420-nonreducing hydrogenase (Mvh) form a large predicted transcription unit; therefore, the Mvh/Hdr/Fwd complex, capable of mediating the electron bifurcation and connecting the first and last steps of methanogenesis, was predicted to be formed in M. conradii. We propose that Methanocella methanogens cope with low H2 and syntrophic growth by (i) stabilizing the Mvh/Hdr/Fwd complex and (ii) activating formatedependent methanogenesis.

Erratum to: Reference genes for transcriptional analysis of flowering and fruit ripening stages in apple (Malus × domestica Borkh.)

Apple (Malus × domestica Borkh.) is the most important deciduous tree fruit crop grown around the world. Comparisons of gene expression profiles from different tissues, conditions or cultivars are valuable scientific tools to better understand the gene expression changes behind important silvicultural and nutritional traits. However, the accuracy of techniques employed to access gene expression is dependent on the evaluation of stable reference genes for data normalization to avoid statistical significance undue or incorrect conclusions. The objective of this work was to select the best genes to be used as references for gene expression studies in apple trees by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Vegetative and reproductive tissues of the apple “Gala” cultivar were evaluated during their seasonal cycle of growth and dormancy. The expression of 23 traditional housekeeping genes or genes suggested as constitutive by microarray data was investigated. Tested combinations of primers allowed the specific amplification and the generation of suitable efficiency curves for gene expression studies by RT-qPCR. Gene stability was determined by two different statistical descriptors, geNorm and NormFinder. The known variable PAL gene expression was used to validate selected normalizers. Results obtained allowed us to conclude that MDH, SAND, THFS, TMp1 and WD40 are the best reference genes to accurately normalize the relative transcript abundances using RT-qPCR in various tissues of apple.

DsrAB-based analysis of sulphate-reducing bacteria in moving bed biofilm reactor (MBBR) wastewater treatment plants.

Sulphate-reducing bacteria (SRB) are important members of the sulphur cycle in wastewater treatment plants (WWTPs). In this study, we investigate the diversity and activity of SRB within the developing and established biofilm of two moving bed biofilm reactor (MBBR) systems treating municipal wastewater in New Zealand. The larger of the two WWTPs (Moa Point) generates high levels of sulphide relative to the smaller Karori plant. Clone libraries of the dissimilatory (bi)sulphite reductase (dsrAB) genes and quantitative real-time PCR targeting dsrA transcripts were used to compare SRB communities between the two WWTPs. Desulfobulbus (35-53% of total SRB sequences) and genera belonging to the family Desulfobacteraceae (27-41%) dominated the SRB fraction of the developing biofilm on deployed plastic carriers at both sites, whereas Desulfovibrio and Desulfomicrobium were exclusively found at Moa Point. In contrast, the established biofilms from resident MBBR carriers were largely dominated by Desulfomonile tiedjei-like organisms (58-100% of SRB sequences). The relative transcript abundance of dsrA genes (signifying active SRBs) increased with biofilm weight yet remained low overall, even in the mature biofilm stage. Our results indicate that although SRB are both present and active in the microbial community at both MBBR study sites, differences in the availability of sulphate may be contributing to the observed differences in sulphide production at these two plants.

Profiling hepatic microRNAs in zebrafish: fluoxetine exposure mimics a fasting response that targets AMP-activated protein kinase (AMPK).

This study examined the similarities in microRNA profiles between fasted and fluoxetine (FLX) exposed zebrafish and downstream target transcripts and biological pathways. Using a custom designed microarray targeting 270 zebrafish miRNAs, we identified 9 differentially expressed miRNAs targeting transcripts in biological pathways associated with anabolic metabolism, such as adipogenesis, cholesterol biosynthesis, triacylglycerol synthesis, and insulin signaling. Exposure of female zebrafish to 540 ng/L FLX, an environmentally relevant concentration and a known metabolic repressor, increased specific miRNAs indicating greater inhibition of these pathways in spite of continued feeding. Further examination revealed two specific miRNAs, dre-let-7d and dre-miR-140-5p, were predicted in silico to bind to a primary regulator of metabolism, adenosine monophosphate-activated protein kinase (AMPK), and more specifically the two isoforms of the catalytic subunit, AMPKα1 and α2, respectively. Real-time analysis of the relative transcript abundance of the α1 and α2 mRNAs indicated a significant inverse relationship between specific miRNA and target transcript. This suggests that AMPK-related pathways may be compromised during FLX exposure as a result of increased miRNA abundance. The mechanism by which FLX regulates miRNA abundance is unknown but may be direct at the liver. The serotonin transporter, slc6a4, is the target of FLX and other selective serotonin reuptake inhibitors (SSRI) and it was found to be expressed in the liver, although treatment did not alter expression of this transporter. Exposure to FLX disrupts key hepatic metabolic pathways, which may be indicative of reduced overall fitness and these effects may be linked to specific miRNA abundance. This has important implications for the heath of fish because concentrations of SSRIs in aquatic ecosystems are continually increasing.

Gene expression and embryo quality in superovulated goats supplemented with crude glycerin after mating

This study aimed to assess the effects of dietary supplementation with crude glycerin on embryo quality and expression profile of genes from superovulated goats. Twenty-four hours (1 day) and 72h (3 days) after mating animals received an oral drench of 200mL saline solution (Control group), 100mL glycerol (100mL group) or 200mL glycerol (200mL group). Both insulin and glucose levels were measured after glycerol drenching. The relative expression of genes (Glut-1, IGF-1, Hsp70, Sod and Bax) in grade I and II embryos from groups was analyzed by sensitive TaqMan qPCR. Administration of crude glycerin raised insulin and glucose (p<0.05) plasma concentrations. Similar rates of superovulatory response (94.12%; 16/17) and recovery of transferable embryos (N embryo grades 1, 2 and 3/N ovulation), (44.69±6.83%; 100/224) were obtained. The 200mL group showed a greater proportion of transferable embryos compared to the degenerated embryos (8.83±2.30 vs. 1.17±0.83, p<0.05), that achieved values 2-fold higher than control group (4.80±2.30 vs. 0.20±0.20, p<0.05) and 100mL group (3.83±1.25 vs. 1.17±0.48, p>0.05). A higher relative abundance of IGF-1 transcripts was observed in 200mL group. Also none differences were observed for transcripts of apoptotic (Bax) and stress (Hsp70) genes. In conclusion, enhancement of circulating glucose and insulin generated from drenching crude glycerin after mating was associated with higher IGF-I expression and increased proportion of transferable embryos.

Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms.

The measurement of gene expression levels in cells and tissues typically depends on a suitable point of reference for inferring biological relevance. For quantitative (or real-time) RT-PCR assays, the method of choice is often to normalize gene expression data to an endogenous gene that is stably expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further recommend that such studies should be accompanied by additional assessment of histology and cellularity of each sample.

Addition of L-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos

The aim of the present study was to determine the effect of L-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification-warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without L-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without L-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with L-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences in TCN, DNA fragmentation and BAX, BCL2L1 and POU5F1 expression were found in vitrified blastocysts among experimental groups. Vitrification procedures increase HSPA1A transcript abundance, but this increase was significantly lower in embryos cultured and/or vitrified with L-ascorbic acid. Thus, supplementing culture and/or vitrification media with L-ascorbic acid enhances survival rates of porcine blastocysts, suggesting a relationship with HSPA1A expression.