Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

The genome sequences of two of the three major species of schistosomes are now available. Molecular tools are needed to determine the importance of these new genes. With this in mind, we investigated introduction of reporter transgenes into schistosome eggs, with the longer-term aim of manipulation of schistosome genes and gene functions. The egg is a desirable developmental stage for genome manipulation, not least because it contains apparently accessible germ cells. Introduction of transgenes into the germ cells of schistosome eggs might result in transgenic schistosomes. However, the egg is surrounded by a thick shell which might block access to entry of transgenes. We cultured eggs in the presence of three types of reporter transgenes of increasing molecular size, and in addition we tried to produce transient holes in the eggs by electroporation to investigate whether the transgenes would more easily enter the eggs. Electroporation of eggs appeared to allow entry of two larger types of transgenes into cultured schistosome eggs, messenger RNA encoding firefly luciferase, and retroviral virions. We anticipate that this approach, electroporation of transgenes into schistosome eggs, will facilitate genetic manipulation of schistosomes for investigating the importance of schistosome genes and gene products as new intervention targets

Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night.

Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption

RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade

The aspartic protease cathepsin D (Clan AA, Family A1) is expressed in the schistosome gut where it plays an apical role in the digestion of hemoglobin released from ingested erythrocytes. In this report, RNA interference approaches were employed to investigate the effects of knockdown of schistosome cathepsin D. Cultured schistosomules of Schistosoma mansoni were exposed by square wave electroporation to double stranded RNA (dsRNA) specific for cDNA encoding S. mansoni cathepsin D. RNAi-mediated reductions in transcript levels led to phenotypic changes including significant growth retardation in vitro and suppression of aspartic protease enzyme activity. In addition, black-pigmented heme, the end point by-product of normal hemoglobin proteolysis that accumulates in the schistosome gut, was not apparent within the guts of the treated schistosomules. Their guts appeared to be red in color, rather than black, apparently indicating the presence of intact rather than digested host hemoglobin. These phenotypic effects were apparent when either of two forms of dsRNA, a long form spanning the entire target transcript or a short form specific for the 3′-region was employed. Off-target effects were not apparent in transcript levels of the gut-localized cysteine protease cathepsin B1. Finally, cathepsin D may be an essential enzyme in the mammal-parasitic stages of schistosomes because schistosomules treated with dsRNA did not survive to maturity after transfer into Balb/c mice. These and earlier findings suggest that, given its essential function in parasite nutrition, schistosome cathepsin D could be developed as a target for novel anti-schistosomal interventions

Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System

<div><p><u>L</u>ong <u>IN</u>terspersed <u>E</u>lement<u>-1</u> (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate <i>cis</i>-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers <i>in vitro</i>. While valuable for understanding ORF1p function, these <i>in vitro</i> approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction <i>in trans</i> leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p <i>cis</i>-preference in L1 mobilization is established, the impact of ability of ORF1pto interact <i>in trans</i> on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.</p> </div

Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing

Abstract Background Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification. Results This method was validated against 16 species of blood-borne helminths and protozoa. Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a substantial reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to undigested samples. This method allowed for discrimination of all common parasitic agents found in human blood, even in cases of multi-parasite infection, and markedly reduced the limit of detection in digested versus undigested samples. Conclusions The results herein provide a novel methodology for the reduction of host DNA prior to TADS and establish the validity of a next-generation sequencing-based platform for universal parasite detection

Analysis of ORF1p interactions <i>in</i><i>trans</i>.

<p><b>A</b>. Schematic of predicted ORF1p species expected to be detected if ORF1p heterodimerization takes place when cells are transiently cotransfected with tagged and untagged ORF1p expression plasmids. Blue rectangle represents ORF1, grey rectangle represents a GAL4 tag, with expected molecular weights expressed in kilodaltons (kDa). Red asterisk denotes heterodimers. <b>B</b>. Western blot analysis of ORF1p species expressed by hORF1co (blue rectangle labeled hORF1co) and hGORF1co (grey/blue rectangle labeled hGORF1co) plasmids individually transfected (hORF1co lane and hGORF1co lane) or cotransfected (hORF1co+hGORF1co lane) in HeLa cells. Western blot analysis of ORF1 protein species in HeLa cells under non-reducing (left panel) and reducing (right panel) conditions. Proteins are detected with human specific polyclonal antibodies. GAPDH used as loading control. A molecular weight ladder in kilodaltons (kDa) is presented on right side. Red asterisk indicates an ORF1p heterodimer. <b>C</b>. The same experiment as in B is performed in NIH-3T3 cells. Non-reducing western blot conditions are shown. Red asterisk denotes ORF1p heterodimers. <b>D</b>. Quantitation of western blot results from B and C using heterodimer/ORF1p monomer ratio. Statistical significance is indicated by dashed lines. An asterisk indicates statistically significant difference (T-test, p-value<0.05).</p

Analysis of subcellular localization of hORF1p expressed from vectors containing wild type sequences.

<p><b>A</b>. (top) Western blot analysis of subcellular localization of ORF1p (blue rectangle labeled as hORF1) expressed from human full-length L1 (hL1wt, 40 kDa), human ORF1 wt (hORF1wt, 41 kDa) or hORF1co (40 kDa) vectors transiently expressed in HeLa cells. Human ORF1p is detected in nuclear (N) and cytoplasmic (C) fractions with human-specific ORF1p polyclonal antibodies. GAPDH (cytoplasmic marker) and Lamin A (nuclear marker) are used as loading and cell fractionation controls. 37 kDa is a molecular marker. Control lanes indicate cells transfected with empty vector. (bottom) Quantitation of western blot results: each construct expressing hORF1p was normalized to its respective GAPDH and Lamin A loading controls. The relative amounts of nuclear and cytoplasmic ORF1p were calculated as a fraction of the relative total ORF1p detected. No statistical significance was found between the subcellular localization of ORF1p expressed by the above-described constructs. <b>B</b>. The same experiment and analysis as in A was performed in NIH-3T3 cells. No statistical significance was found between the subcellular localization of ORF1p expressed by the above-described constructs. <b>C</b>. Subcellular localization of mORF1p (green rectangle labeled as mORF1) transiently expressed from the full-length mouse L1 (mL1wt) or codon-optimized mORF1 (mORF1co) in HeLa or NIH-3T3 cells using mouse specific polyclonal antibodies. 50 kDa is a molecular marker. </p