BackgroundThe ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation.Methodology/Principal FindingsThis RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data.Conclusions/SignificanceRNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.

The intestinal barrier integrity is essential for the absorption of nutrients and health in humans and animals. Dysfunction of the mucosal barrier is associated with increased gut permeability and development of multiple gastrointestinal diseases. Recent studies highlighted a critical role for glutamine, which had been traditionally considered as a nutritionally non-essential amino acid, in activating the mammalian target of rapamycin cell signaling in enterocytes. In addition, glutamine has been reported to enhance intestinal and whole-body growth, to promote enterocyte proliferation and survival, and to regulate intestinal barrier function in injury, infection, weaning stress, and other catabolic conditions. Mechanistically, these effects were mediated by maintaining the intracellular redox status and regulating expression of genes associated with various signaling pathways. Furthermore, glutamine stimulates growth of the small intestinal mucosa in young animals and also enhances ion transport by the gut in neonates and adults. Growing evidence supports the notion that glutamine is a nutritionally essential amino acid for neonates and a conditionally essential amino acid for adults. Thus, as a functional amino acid with multiple key physiological roles, glutamine holds great promise in protecting the gut from atrophy and injury under various stress conditions in mammals and other animals.

Members of Myxozoa, a parasitic metazoan taxon, have considerable detrimental effects on fish hosts and also have been associated with human food-borne illness. Little is known about their biology and metabolism. Analysis of the genome of Thelohanellus kitauei and comparative analysis with genomes of its two free-living cnidarian relatives revealed that T. kitauei has adapted to parasitism, as indicated by the streamlined metabolic repertoire and the tendency toward anabolism rather than catabolism. Thelohanellus kitauei mainly secretes proteases and protease inhibitors for nutrient digestion (parasite invasion), and depends on endocytosis (mainly low-density lipoprotein receptors-mediated type) and secondary carriers for nutrient absorption. Absence of both classic and complementary anaerobic pathways and gluconeogenesis, the lack of de novo synthesis and reduced activity in hydrolysis of fatty acids, amino acids, and nucleotides indicated that T. kitauei in this vertebrate host–parasite system has adapted to inhabit a physiological environment extremely rich in both oxygen and nutrients (especially glucose), which is consistent with its preferred parasitic site, that is, the host gut submucosa. Taking advantage of the genomic and transcriptomic information, 23 potential nutrition-related T. kitauei-specific chemotherapeutic targets were identified. This first genome sequence of a myxozoan will facilitate development of potential therapeutics for efficient control of myxozoan parasites and ultimately prevent myxozoan-induced fish-borne illnesses in humans.

Apple ring rot and Botryosphaeria canker are severe diseases affecting apple production in China, but there is confusion regarding which pathogens cause these diseases and their similarity to other diseases, such as white rot of apple, and ring rot and Botryosphaeria canker of pear. In this study, the pathogen of apple ring rot in China was compared with the pathogen of apple ring rot in Japan and Korea, the pathogen of Botryosphaeria canker of apple and pear in China, the pathogen of pear ring rot in China, and the pathogen of white rot of apple in the United States. Comparisons were based on morphology, pathogenicity on branches and fruit, and sequences of rDNA in the internal transcribed spacer region and of the β-tubulin and actin genes. Results showed that the causal agent of apple ring rot and Botryosphaeria canker of apple in China was Botryosphaeria dothidea, which has also been reported to be the pathogen of apple ring rot in Korea and Japan. Pathogenicity tests showed that B. dothidea infection on apple and pear branches may induce wart or canker symptoms depending on the conditions. These results are consistent with the hypothesis that the same pathogen causes the wart symptom of apple ring rot and the Botryosphaeria canker symptom on apple branches in China. The results also suggest that apple ring rot and white rot are the same disease and are caused by B. dothidea. Finally, B. dothidea isolates from pear and other fruit or forest trees may serve as inoculum for apple ring rot.

Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone, and they have been implicated in axon guidance; however, the detailed localization, trafficking, and cellular functions of Src kinases in live growth cones are unclear. Here, we cloned two novel Aplysia Src kinases, termed Src1 and Src2, and we show their association with both the plasma membrane and the microtubule cytoskeleton in the growth cone by live cell imaging, immunocytochemistry, and cell fractionation. Activated Src2 is enriched in filopodia tips. Interestingly, Src2-enhanced green fluorescent protein-positive endocytic vesicles and tubulovesicular structures undergo microtubule-mediated movements that are bidirectional in the central domain and mainly retrograde in the peripheral domain. To further test the role of microtubules in Src trafficking in the growth cone, microtubules were depleted with either nocodazole or vinblastine treatment, resulting in an increase in Src2 plasma membrane levels in all growth cone domains. Our data suggest that microtubules regulate the steady-state level of active Src at the plasma membrane by mediating retrograde recycling of endocytosed Src. Expression of constitutively active Src2 results in longer filopodia that protrude from smaller growth cones, implicating Src2 in controlling the size of filopodia and lamellipodia.

A xylanase-encoding gene, designated xynA19, was cloned from Sphingobacterium sp. TN19--a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae--and expressed in Escherichia coli BL21 (DE3). The full-length xynA19 (1,155 bp in length) encodes a 384-residue polypeptide (XynA19) containing a predicted signal peptide of 24 residues and a catalytic domain belonging to glycosyl hydrolase family 10 (GH 10). The deduced amino acid sequence of XynA19 is most similar (53.1% identity) to an endo-1,4-beta-xylanase from Prevotella bryantii B(1)4. Phylogenetic analysis of GH 10 Bacteroidia xylanases indicated that GH 10 xylanases from Sphingobacteria were separated into two clusters, and XynA19 is more closely related to the xylanases of Bacteroidia from gut or rumen than to those of Flavobacteria and Sphingobacteria from other sources. Recombinant XynA19 (r-XynA19) showed apparent optimal activity at pH 6.5 and 45 degrees C. Compared with thermophilic and mesophilic counterparts, r-XynA19 was more active at low temperatures, retaining >65% of its maximum activity at 20-28 degrees C and approximately 40% even at 10 degrees C, and modeling indicated that XynA19 has fewer hydrogen bonds and salt bridges. These properties suggest that XynA19 has various potential applications, especially in aquaculture and the food industry.

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.

A phytase with high activity at neutral pH and typical water temperatures ( approximately 25 degrees C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity ( approximately 28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg(-1). The optimum pH was 7.0, and the optimum temperature was 45 degrees C. The K (m), V (max), and k (cat) values were 1.28 mM, 71.9 micromol min(-1) mg(-1), and 45.1 s(-1), respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25 degrees C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.