Comparative transcriptome sequencing of germline and somatic tissues of the Ascaris suum gonad

<p>Abstract</p> <p>Background</p> <p><it>Ascaris suum </it>(large roundworm of pigs) is a parasitic nematode that causes substantial losses to the meat industry. This nematode is suitable for biochemical studies because, unlike <it>C. elegans</it>, homogeneous tissue samples can be obtained by dissection. It has large sperm, produced in great numbers that permit biochemical studies of sperm motility. Widespread study of <it>A. suum </it>would be facilitated by more comprehensive genome resources and, to this end, we have produced a gonad transcriptome of <it>A. suum</it>.</p> <p>Results</p> <p>Two 454 pyrosequencing runs generated 572,982 and 588,651 reads for germline (TES) and somatic (VAS) tissues of the <it>A. suum </it>gonad, respectively. 86% of the high-quality (HQ) reads were assembled into 9,955 contigs and 69,791 HQ reads remained as singletons. 2.4 million bp of unique sequences were obtained with a coverage that reached 16.1-fold. 4,877 contigs and 14,339 singletons were annotated according to the <it>C. elegans </it>protein and the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases. Comparison of TES and VAS transcriptomes demonstrated that genes participating in DNA replication, RNA transcription and ubiquitin-proteasome pathways are expressed at significantly higher levels in TES tissues than in VAS tissues. Comparison of the <it>A. suum </it>TES transcriptome with the <it>C. elegans </it>microarray dataset identified 165 <it>A. suum </it>germline-enriched genes (83% are spermatogenesis-enriched). Many of these genes encode serine/threonine kinases and phosphatases (KPs) as well as tyrosine KPs. Immunoblot analysis further suggested a critical role of phosphorylation in both testis development and spermatogenesis. A total of 2,681 <it>A. suum </it>genes were identified to have associated RNAi phenotypes in <it>C. elegans</it>, the majority of which display embryonic lethality, slow growth, larval arrest or sterility.</p> <p>Conclusions</p> <p>Using deep sequencing technology, this study has produced a gonad transcriptome of <it>A. suum</it>. By comparison with <it>C. elegans </it>datasets, we identified sets of genes associated with spermatogenesis and gonad development in <it>A. suum</it>. The newly identified genes encoding KPs may help determine signaling pathways that operate during spermatogenesis. A large portion of <it>A. suum </it>gonadal genes have related RNAi phenotypes in <it>C. elegans </it>and, thus, might be RNAi targets for parasite control.</p

Developmental Transcriptomic Features of the Carcinogenic Liver Fluke, Clonorchis sinensis

Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens

Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC50 values ranging from 6–9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71

Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells.

LCK is a tyrosine kinase that is essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active-site lysine is replaced by a photocaged equivalent, using genetic code expansion. This strategy enabled fine temporal and spatial control over kinase activity, thus allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that autophosphorylation of the LCK active-site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, T-cell coreceptors, can enhance LCK activity, thereby helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics