A Quantitative PCR System for Measuring Sclerotinia sclerotiorum in Canola (Brassica napus).

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is an economically important disease of canola (Brassica napus) commonly managed by routine application of fungicides. Petal infestation has been demonstrated to be an important stage of the disease cycle in canola and has been the focus of previously developed Sclerotinia stem rot risk assessment methods. Quantitative polymerase chain reaction (qPCR) analysis can provide a more rapid and accurate assessment of petal infestation levels. Primers and a hydrolysis probe were designed to amplify a 70-bp region of an S. sclerotiorum-specific gene, SS1G_00263. A hydrolysis probe-based qPCR assay was developed that had a detection limit of 8.0 × 10-4 ng of S. sclerotiorum DNA and only amplified S. sclerotiorum DNA. Evaluation of petals collected at five sampling points in each of 10 commercial canola fields on each of two sampling dates (corresponding to 20 to 30% bloom and 40 to 50% bloom) revealed S. sclerotiorum DNA infestation levels of 0 to 3.3 × 10-1 ng/petal. This qPCR assay can be used to reliably quantify petal infestation and, with further research, has the potential to serve as the basis for a Sclerotinia stem rot risk assessment tool or as a means to study Sclerotinia stem rot epidemiology.

Microarray analysis in caudal medulla of cattle orally challenged with bovine spongiform encephalopathy.

Bovine spongiform encephalopathy (BSE) is a fatal disorder in cattle characterized by progressive neurodegeneration of the central nervous system. We investigated the molecular mechanisms involved in neurodegeneration during prion infection through the identification of genes that are differentially expressed (DE) between experimentally infected and non-challenged cattle. Gene expression of caudal medulla from control and orally infected animals was compared by microarray analysis using 24,000 bovine oligonucleotides representing 16,846 different genes to identify DE genes associated with BSE disease. In total, 182 DE genes were identified between normal and BSE-infected tissues (>2.0-fold change, P < 0.01); 81 DE genes had gene ontology functions, which included synapse function, calcium ion regulation, immune and inflammatory response, apoptosis, and cytoskeleton organization; 13 of these genes were found to be involved in 26 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression of five DE genes associated with synapse function (tachykinin, synuclein, neuropeptide Y, cocaine, amphetamine-responsive transcript, and synaptosomal-associated protein 25 kDa) and three DE genes associated with calcium ion regulation (parvalbumin, visinin-like, and cadherin) was further validated in the medulla tissue of cattle at different infection times (6, 12, 42, and 45 months post-infection) by qRT-PCR. These data will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species.

The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis.

Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. In Saccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.

Astronomy. ASASSN-15lh: A highly super-luminous supernova.

We report the discovery of ASASSN-15lh (SN 2015L), which we interpret as the most luminous supernova yet found. At redshift z = 0.2326, ASASSN-15lh reached an absolute magnitude of Mu ,AB = -23.5 ± 0.1 and bolometric luminosity Lbol = (2.2 ± 0.2) × 10(45) ergs s(-1), which is more than twice as luminous as any previously known supernova. It has several major features characteristic of the hydrogen-poor super-luminous supernovae (SLSNe-I), whose energy sources and progenitors are currently poorly understood. In contrast to most previously known SLSNe-I that reside in star-forming dwarf galaxies, ASASSN-15lh appears to be hosted by a luminous galaxy (MK ≈ -25.5) with little star formation. In the 4 months since first detection, ASASSN-15lh radiated (1.1 ± 0.2) × 10(52) ergs, challenging the magnetar model for its engine.

Induction of Microsomal Membrane Proteins in Roots of an Aluminum-Resistant Cultivar of Triticum aestivum L. under Conditions of Aluminum Stress.

Three-day-old seedlings of an Al-sensitive (Neepawa) and an Al-resistant (PT741) cultivar of Triticum aestivum were subjected to Al concentrations ranging from 0 to 100 [mu]M for 72 h. At 25 [mu]M Al, growth of roots was inhibited by 57% in the Al-sensitive cultivar, whereas root growth in the Al-resistant cultivar was unaffected. A concentration of 100 [mu]M Al was required to inhibit root growth of the Al-resistant cultivar by 50% and resulted in almost total inhibition of root growth in the sensitive cultivar. Cytoplasmic and microsomal membrane fractions were isolated from root tips (first 5 mm) and the adjacent 2-cm region of roots of both cultivars. When root cytoplasmic proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no changes in polypeptide patterns were observed in response to Al stress. Analysis of microsomal membrane proteins revealed a band with an apparent molecular mass of 51 kD, which showed significant accumulation in the resistant cultivar following Al exposure. Two-dimensional gel analysis revealed that this band comprises two polypeptides, each of which is induced by exposure to Al. The response of the 51-kD band to a variety of experimental conditions was characterized to determine whether its pattern of accumulation was consistent with a possible role in Al resistance. Accumulation was significantly greater in root tips when compared to the rest of the root. When seedlings were subjected to Al concentrations ranging from 0 to 150 [mu]M, the proteins were evident at 25 [mu]M and were fully accumulated at 100 [mu]M. Time-course studies from 0 to 96 h indicated that full accumulation of the 51-kD band occurred within 24 h of initiation of Al stress. With subsequent removal of stress, the polypeptides gradually disappeared and were no longer visible after 72 h. When protein synthesis was inhibited by cycloheximide, the 51-kD band disappeared even when seedlings were maintained in Al-containing media. Other metals, including Cu, Zn, and Mn, failed to induce this band, and Cd and Ni resulted in its partial accumulation. These results indicate that synthesis of the 51-kD microsomal membrane proteins is specifically induced and maintained during Al stress in the Al-resistant cultivar, PT741.

Differential Exudation of Polypeptides by Roots of Aluminum-Resistant and Aluminum-Sensitive Cultivars of Triticum aestivum L. in Response to Aluminum Stress.

Cultivars of Triticum aestivum differing in resistance to Al were grown under aseptic conditions in the presence and absence of Al and polypeptides present in root exudates were collected, concentrated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon exposure to 100 and 200 [mu]M Al, root elongation in Al-sensitive cultivars was reduced by 30 and 65%, respectively, whereas root elongation in resistant cultivars was reduced by only 15 and 30%. Accumulation of polypeptides in the growth medium increased with time for 96 to 120 h, with little additional accumulation thereafter. This pattern of exudation was virtually unaffected by exposure to 100 [mu]M Al in the Al-resistant cultivars Atlas 66 and Maringa, whereas total accumulation was reduced in sensitive cultivars. Changes in exudation were consistent with alterations in root elongation. Al-induced or Al-enhanced polypeptide bands were detected in Atlas 66 and Maringa after 72 h of exposure to Al. Increased accumulation of 12-, 22-, and 33-kD bands was observed at 75 [mu]M Al in Atlas 66 and 12-, 23-, and 43.5-kD bands started to appear at 50 [mu]M Al in Maringa. In the Al-sensitive cultivars Roblin and Katepwa, no significant effect on polypeptide profiles was observed at values up to 100 [mu]M Al. When root exudates were separated by ultrafiltration and the Al content was measured in both high molecular mass (HMM; >10 kD) and ultrafiltrate (<10 kD) fractions, approximately 2 times more Al was detected in HMM fractions from Al-resistant cultivars than from Al-sensitive cultivars. Dialysis of HMM fractions against water did not release this bound Al;digestion with protease released between 62 and 73% of total Al, with twice as much released from exudates of Al-resistant than of Al-sensitive cultivars. When plants were grown in the presence of 0 to 200 [mu]M Al, saturation of the Al-binding capacity of HMM exudates occurred at 50 [mu]M Al in Al-sensitive cultivars. Saturation was not achieved in resistant cultivars. Differences in exudation of total polypeptides in response to Al stress, enhanced accumulation of specific polypeptides, and the greater association of Al with HMM fractions from Al-resistant cultivars suggest that root exudate polypeptides may play a role in plant response to Al.

Al-Induced, 51-Kilodalton, Membrane-Bound Proteins Are Associated with Resistance to Al in a Segregating Population of Wheat.

Incorporation of 35S into protein is reduced by exposure to Al in wheat (Triticum aestivum), but the effects are genotype-specific. Exposure to 10 to 75 [mu]M Al had little effect on 35S incorporation into total protein, nuclear and mitochondrial protein, microsomal protein, and cytosolic protein in the Al-resistant cultivar PT741. In contrast, 10 [mu]M Al reduced incorporation by 21 to 38% in the Al-sensitive cultivar Katepwa, with effects becoming more pronounced (31-62%) as concentrations of Al increased. We previously reported that a pair of 51-kD membrane-bound proteins accumulated in root tips of PT741 under conditions of Al stress. We now report that the 51-kD band is labeled with 35S after 24 h of exposure to 75 [mu]M Al. The specific induction of the 51-kD band in PT741 suggested a potential role of one or both of these proteins in mediating resistance to Al. Therefore, we analyzed their expression in single plants from an F2 population arising from a cross between the PT741 and Katepwa cultivars. Accumulation of 1,3-[beta]-glucans (callose) in root tips after 24 h of exposure to 100 [mu]M Al indicated that this population segregated for Al resistance in about a 3:1 ratio. A close correlation between resistance to Al (low callose content of root tips) and accumulation of the 51-kD band was observed, indicating that at least one of these proteins cosegregates with the Al-resistance phenotype. As a first step in identifying a possible function, we have demonstrated that the 51-kD band is most clearly associated with the tonoplast. Whereas Al has been reported to stimulate the activity of the tonoplast H+-ATPase and H+-PPase, antibodies raised against these proteins did not cross-react with the 51-kD band. Efforts are now under way to purify this protein from tonoplast-enriched fractions.

Preadipocyte and adipose tissue differentiation in meat animals: influence of species and anatomical location.

Early in porcine adipose tissue development, the stromal-vascular (SV) elements control and dictate the extent of adipogenesis in a depot-dependent manner. The vasculature and collagen matrix differentiate before overt adipocyte differentiation. In the fetal pig, subcutaneous (SQ) layer development is predictive of adipocyte development, as the outer, middle, and inner layers of dorsal SQ adipose tissue develop and maintain layered morphology throughout postnatal growth of SQ adipose tissue. Bovine and ovine fetuses contain brown adipose tissue but SQ white adipose tissue is poorly developed structurally. Fetal adipose tissue differentiation is associated with the precocious expression of several genes encoding secreted factors and key transcription factors like peroxisome proliferator activated receptor (PPAR)γ and CCAAT/-enhancer-binding protein. Identification of adipocyte-associated genes differentially expressed by age, depot, and species in vivo and in vitro has been achieved using single-gene analysis, microarrays, suppressive subtraction hybridization, and next-generation sequencing applications. Gene polymorphisms in PPARγ, cathepsins, and uncoupling protein 3 have been associated with back fat accumulation. Genome scans have mapped several quantitative trait loci (QTL) predictive of adipose tissue-deposition phenotypes in cattle and pigs.

Transcriptome analysis of subcutaneous adipose tissues in beef cattle using 3' digital gene expression-tag profiling.

The molecular mechanisms that regulate fat deposition in bovine adipose tissue have not been well studied. To elucidate the genes and gene networks involved in bovine fat development, transcriptional profiles of backfat (BF) tissues from Hereford × Aberdeen Angus (HEAN, n = 6) and Charolais × Red Angus (CHRA, n = 6) steers with high or low BF thickness were characterized by digital gene expression-tag profiling. Approximately 9.8 to 21.9 million tags were obtained for each library, and a total of 18,034 genes were identified. In total, 650 genes were found to be differentially expressed, with a greater than 1.5-fold difference between the 2 crossbreds (Benjamini-Hochberg false discovery rate ≤ 0.05). The majority of differentially expressed genes that were more highly expressed in CHRA vs. HEAN were associated with development, whereas the differentially expressed genes with greater expression in HEAN vs. CHRA were overrepresented in biological processes such as metabolism and immune response. Thirty-six and 152 differentially expressed genes were detected between animals with high (n = 3) and low (n = 3) BF thickness in HEAN and CHRA, respectively (Benjamini-Hochberg false discovery rate ≤0.05). The differentially expressed genes between high and low groups in CHRA were related to cell proliferation and development processes. In addition, lipid metabolism was 1 of the top 5 molecular and cellular functions identified in both crossbreds. Ten and 17 differentially expressed genes were found to be involved in fat metabolism in HEAN and CHRA, respectively. Genes associated with obesity, such as PTX3 (pentraxin 3, long) and SERPINE1 (serpin peptidase inhibitor, clade E, member 1), were more highly expressed (P < 0.05) in the subset of CHRA animals with greater BF thickness. Our study revealed that the expression patterns of genes in BF tissues differed depending on the genetic background of the cattle.

Differential sensitivity to inhibitors discriminates between two types of kinases responsible for in vivo phosphorylation of different sites in the carboxy-terminal tail of chicken neurofilament-M.

In order to characterize the phosphorylation of neurofilaments (NF) in intact neurons, we examined the ability of several protein kinase inhibitors to interfere with the incorporation 32P into individual NF polypeptides of sensory neurons in culture. We also examined their effect on the post-translational mobility shift on SDS-PAGE that accompanies phosphorylation of newly synthesized NF-M. Several agents known to inhibit cyclic nucleotide-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases (H7, HA1004, trifluoperizine, sphingosine) had no effect on the phosphorylation of any NF polypeptide, in either assay. In contrast, two broadly active protein kinase inhibitors, staurosporine and K252a, inhibited the incorporation of 32P into NF-M by 60-70% and also blocked the post-translational mobility shift. They had no effect on NF-L. The action of staurosporine and K252a was identical to that of 25 mM LiCl. Proteolytic cleavage and phosphopeptide mapping of 32P-labeled NF-M from control and treated cultures revealed that the phosphorylation of only one subset of phosphopeptides was affected by staurosporine, K252a, and LiCl. These were contained within a single chymotryptic fragment of the NF-M tail segment, probably containing most of the 17 repeats of a KXXS/TP motif. The phosphorylation of another subset of phosphopeptides was insensitive to these inhibitors. They were contained within a different chymotryptic fragment of the tail segment which contains a KSD and four KSP potential phosphorylation sites. This differential sensitivity to protein kinase inhibitors distinguishes two different types of effector-independent kinases that phosphorylate, in vivo, different sites within the NF-M tail.

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of PHGPX and URE2 genes in aluminum resistance in Saccharomyces cerevisiae.

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 Delta, phgpx2 Delta, and phgpx3Delta), were tested for their sensitivity to Al. The triple mutant, phgpx1 Delta/2Delta/3Delta, was more sensitive to Al (55% reduction in growth at 300 microM Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3Delta to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3Delta and phgpx1Delta/2Delta/3Delta mutants were sensitive to oxidative stress (exposure to H(2)O(2) or diamide). Lipid peroxidation was also increased in the phgpx1Delta/2Delta/3Delta mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1Delta and gtt2Delta), glutathione biosynthesis (gsh1Delta and gsh2Delta), glutathione reductase (glr1Delta) and a glutathione transporter (opt1Delta) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2Delta mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 microM Al. Intracellular oxidation levels in the ure2Delta mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H(2)O(2)) increase compared to BY4741; however, the ure2Delta mutant showed no change in lipid peroxidation compared to the control. The phgpx1Delta/2Delta/3Delta and ure2Delta mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.