Organ-, sex- and age-dependent patterns of endogenous L1 mRNA expression at a single locus resolution.

Expression of L1 mRNA, the first step in the L1 copy-and-paste amplification cycle, is a prerequisite for L1-associated genomic instability. We used a reported stringent bioinformatics method to parse L1 mRNA transcripts and measure the level of L1 mRNA expressed in mouse and rat organs at a locus-specific resolution. This analysis determined that mRNA expression of L1 loci in rodents exhibits striking organ specificity with less than 0.8% of loci shared between organs of the same organism. This organ specificity in L1 mRNA expression is preserved in male and female mice and across age groups. We discovered notable differences in L1 mRNA expression between sexes with only 5% of expressed L1 loci shared between male and female mice. Moreover, we report that the levels of total L1 mRNA expression and the number and spectrum of expressed L1 loci fluctuate with age as independent variables, demonstrating different patterns in different organs and sexes. Overall, our comparisons between organs and sexes and across ages ranging from 2 to 22 months establish previously unforeseen dynamic changes in L1 mRNA expression in vivo. These findings establish the beginning of an atlas of endogenous L1 mRNA expression across a broad range of biological variables that will guide future studies.

Altered DNA repair creates novel Alu/Alu repeat-mediated deletions.

Alu elements are the most abundant source of nonallelic homology that influences genetic instability in the human genome. When there is a DNA double-stranded break, the Alu element's high copy number, moderate length and distance and mismatch between elements uniquely influence recombination processes. We utilize a reporter-gene assay to show the complex influence of Alu mismatches on Alu-related repeat-mediated deletions (RMDs). The Alu/Alu heteroduplex intermediate can result in a nonallelic homologous recombination (HR). Alternatively, the heteroduplex can result in various DNA breaks around the Alu elements caused by competing nucleases. These breaks can undergo Alt-nonhomologous end joining to cause deletions focused around the Alu elements. Formation of these heteroduplex intermediates is largely RAD52 dependent. Cells with low ERCC1 levels utilize more of these alternatives resolutions, while cells with MSH2 defects tend to have more RMDs with a specific increase in the HR events. Therefore, Alu elements are expected to create different forms of deletions in various cancers depending on a number of these DNA repair defects.

Long-Distance Relationships: Suppression of Repeat-Mediated Deletions.

The high proportion of repetitive DNA sequences in the human genome provides tremendous opportunities for DNA rearrangements between non-allelic repetitive elements. The genome must use multiple competing and collaborating repair mechanisms to minimize these types of DNA rearrangements, some of which fail in cancer cells where DNA repair pathways are suppressed.

A comprehensive approach to expression of L1 loci.

L1 elements represent the only currently active, autonomous retrotransposon in the human genome, and they make major contributions to human genetic instability. The vast majority of the 500 000 L1 elements in the genome are defective, and only a relatively few can contribute to the retrotransposition process. However, there is currently no comprehensive approach to identify the specific loci that are actively transcribed separate from the excess of L1-related sequences that are co-transcribed within genes. We have developed RNA-Seq procedures, as well as a 1200 bp 5΄ RACE product coupled with PACBio sequencing that can identify the specific L1 loci that contribute most of the L1-related RNA reads. At least 99% of L1-related sequences found in RNA do not arise from the L1 promoter, instead representing pieces of L1 incorporated in other cellular RNAs. In any given cell type a relatively few active L1 loci contribute to the 'authentic' L1 transcripts that arise from the L1 promoter, with significantly different loci seen expressed in different tissues.

The contribution of alu elements to mutagenic DNA double-strand break repair.

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

Culture for genetic manipulation of developmental stages of Schistosoma mansoni.

Genomes of the major human helminth parasites, and indeed many others of agricultural significance, are now the research focus of intensive genome sequencing and annotation. A draft genome sequence of the filarial parasite Brugia malayi was reported in 2007 and draft genomes of two of the human schistosomes, Schistosoma japonicum and S. mansoni reported in 2009. These genome data provide the basis for a comprehensive understanding of the molecular mechanisms involved in schistosome nutrition and metabolism, host-dependent development and maturation, immune evasion and invertebrate evolution. In addition, new potential vaccine candidates and drug targets will likely be predicted. However, testing these predictions is often not straightforward with schistosomes because of the difficulty and expense in maintenance of the developmental cycle. To facilitate this goal, several developmental stages can be maintained in vitro for shorter or longer intervals of time, and these are amenable to manipulation. Our research interests focus on experimental studies of schistosome gene functions, and more recently have focused on development of transgenesis and RNA interference with the longer term aim of heritable gene manipulation. Here we review methods to isolate and culture developmental stages of Schistosoma mansoni, including eggs, sporocysts, schistosomules and adults, in particular as these procedures relate to approaches for gene manipulation. We also discuss recent advances in genetic manipulation of schistosomes including the deployment of square wave electroporation to introduce reporter genes into cultured schistosomes.

Comparative analysis on the expression of L1 loci using various RNA-Seq preparations.

BACKGROUND: Retrotransposons are one of the oldest evolutionary forces shaping mammalian genomes, with the ability to mobilize from one genomic location to another. This mobilization is also a significant factor in human disease. The only autonomous human retroelement, L1, has propagated to make up 17% of the human genome, accumulating over 500,000 copies. The majority of these loci are truncated or defective with only a few reported to remain capable of retrotransposition. We have previously published a strand-specific RNA-Seq bioinformatics approach to stringently identify at the locus-specific level the few expressed full-length L1s using cytoplasmic RNA. With growing repositories of RNA-Seq data, there is potential to mine these datasets to identify and study expressed L1s at single-locus resolution, although many datasets are not strand-specific or not generated from cytoplasmic RNA. RESULTS: We developed whole-cell, cytoplasmic and nuclear RNA-Seq datasets from 22Rv1 prostate cancer cells to test the influence of different preparations on the quality and effort needed to measure L1 expression. We found that there was minimal data loss in the identification of full-length expressed L1 s using whole cell, strand-specific RNA-Seq data compared to cytoplasmic, strand-specific RNA-Seq data. However, this was only possible with an increased amount of manual curation of the bioinformatics output to eliminate increased background. About half of the data was lost when the sequenced datasets were non-strand specific. CONCLUSIONS: The results of these studies demonstrate that with rigorous manual curation the utilization of stranded RNA-Seq datasets allow identification of expressed L1 loci from either cytoplasmic or whole-cell RNA-Seq datasets.

Postharvest heat treatments to inhibit Penicillium digitatum growth and maintain quality of Mandarin (Citrus reticulata blanco).

Use of fungicides is a common practice as a postharvest treatment to control fruit decay. Nowadays, environment friendly technologies, such as heat treatments, are viable replacements. This study evaluated the effects of post-harvest heat treatments (traditional and microwave-assisted) on mandarins intentionally inoculated with Penicillium digitatum. For the studied heat treatments, the target temperature was 50 °C, which was held for 2.5 min. After heating, mandarins were cooled and stored at 25 °C for 13 days. MW treatments effectively prevented mold growth during storage, while HW only delayed it. Control mandarins (without treatment) showed the highest significant weight loss. Neither thermal treatment nor storage affected fruit juice pH (p > 0.05). Treated mandarins had a significantly lower vitamin C content than control fruits throughout storage, and all mandarins lost firmness by the 13th day (p < 0.05). Control and MW-treated mandarins had lower citric acid content; however, they retained color, total soluble solids (TSS) and had a higher maturity index. While HW mandarins did not have changes in citric acid content, they had higher TSS, and lower maturity index. MW-assisted treatments were effective at inactivating molds and helped retain some nutritional and physical-chemical characteristics of mandarins. However, juice of MW-treated mandarins was not preferred by judges in the sensory tests, the juice was rated lower than that obtained from the other treatment. Postharvest heat treatments may constitute a helpful application to control mandarin' fungal decay.

Integration of reporter transgenes into Schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus.

The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus-mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG-pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR-based approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward toward germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes.

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level.

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and mutagenesis. Understanding the expression patterns of L1 loci at the individual level will lend to the understanding of the biology of this mutagenic element. This autonomous element makes up a significant portion of the human genome with over 500,000 copies, though 99% are truncated and defective. However, their abundance and dominant number of defective copies make it challenging to identify authentically expressed L1s from L1-related sequences expressed as part of other genes. It is also challenging to identify which specific L1 locus is expressed due to the repetitive nature of the elements. Overcoming these challenges, we present an RNA-Seq bioinformatic approach to identify L1 expression at the locus specific level. In summary, we collect cytoplasmic RNA, select for polyadenylated transcripts, and utilize strand-specific RNA-Seq analyses to uniquely map reads to L1 loci in the human reference genome. We visually curate each L1 locus with uniquely mapped reads to confirm transcription from its own promoter and adjust mapped transcript reads to account for mappability of each individual L1 locus. This approach was applied to a prostate tumor cell line, DU145, to demonstrate the ability of this protocol to detect expression from a small number of the full-length L1 elements.

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.

piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni.

The transposon piggyBac from the genome of the cabbage looper moth Trichoplusia ni has been observed in the laboratory to jump into the genomes of key model and pathogenic eukaryote organisms including mosquitoes, planarians, human and other mammalian cells, and the malaria parasite Plasmodium falciparum. Introduction of exogenous transposons into schistosomes has not been reported but transposon-mediated transgenesis of schistosomes might supersede current methods for functional genomics of this important human pathogen. In the present study we examined whether the piggyBac transposon could deliver reporter transgenes into the genome of Schistosoma mansoni parasites. A piggyBac donor plasmid modified to encode firefly luciferase under control of schistosome gene promoters was introduced along with 7-methylguanosine capped RNAs encoding piggyBac transposase into cultured schistosomula by square wave electroporation. The activity of the helper transposase mRNA was confirmed by Southern hybridization analysis of genomic DNA from the transformed schistosomes, and hybridization signals indicated that the piggyBac transposon had integrated into numerous sites within the parasite chromosomes. piggyBac integrations were recovered by retrotransposon-anchored PCR, revealing characteristic piggyBac TTAA footprints in the vicinity of the endogenous schistosome retrotransposons Boudicca, SR1, and SR2. This is the first report of chromosomal integration of a transgene and somatic transgenesis of this important human pathogen, in this instance accomplished by mobilization of the piggyBac transposon.

Serial Procalcitonin as a Predictor of Bacteremia and Need for Intensive Care Unit Care in Adults With Pneumonia, Including Those With Highest Severity: A Prospective Cohort Study.

BACKGROUND: Procalcitonin (PCT) is a prohormone that rises in bacterial pneumonia and has promise in reducing antibiotic use. Despite these attributes, there are inconclusive data on its use for clinical prognostication. We hypothesize that serial PCT measurements can predict mortality, intensive care unit (ICU) admission, and bacteremia. METHODS: A prospective cohort study of inpatients diagnosed with pneumonia was performed at a large tertiary care center in Boston, Massachusetts. Procalcitonin was measured on days 1 through 4. The primary endpoint was a composite adverse outcome defined as all-cause mortality, ICU admission, and bacteremia. Regression models were calculated with area under the receiver operating characteristic curve (AUC) as a measure of discrimination. RESULTS: Of 505 patients, 317 patients had a final diagnosis of community-acquired pneumonia (CAP) or healthcare-associated pneumonia (HCAP). Procalcitonin was significantly higher for CAP and HCAP patients meeting the composite primary endpoint, bacteremia, and ICU admission, but not mortality. Incorporation of serial PCT levels into a statistical model including the Pneumonia Severity Index (PSI) improved the prognostic performance of the PSI with respect to the primary composite endpoint (AUC from 0.61 to 0.66), bacteremia (AUC from 0.67 to 0.85), and need for ICU-level care (AUC from 0.58 to 0.64). For patients in the highest risk class PSI >130, PCT was capable of further risk stratification for prediction of adverse outcomes. CONCLUSION: Serial PCT measurement in patients with pneumonia shows promise for predicting adverse clinical outcomes, including in those at highest mortality risk.

Organization and functional analysis of the Schistosoma mansoni cathepsin D-like aspartic protease gene promoter.

We have cloned a 969-bp fragment of genomic DNA that spans 821 bp of the 5' untranslated region, exon 1, a short intron, and part of exon 2 of the Schistosoma mansoni cathepsin D gene by inverse PCR. Inspection of this sequence revealed the presence of two TATA-box motifs, two inverted CCAAT-box (inverted NF-Y) motifs and sequences with homology to binding sites for the transcription factors, AP-1 and NF-Y. This sequence and deletion variants were cloned into reporter gene constructs, in order to examine the ability of these putative regulatory sequences to drive heterologous reporter gene activity. PCR products were cloned into the luciferase reporter vector pXP2. These reporter gene constructs were used to transform HeLa cells which were cultured and examined for luciferase activity. Additionally, HeLa cells transiently transfected with an EGFP reporter plasmid driven by the putative promoter from the S. mansoni cathepsin D gene were examined for EGFP transcripts and fluorescence. The 5' untranslated region of the S. mansoni cathepsin D gene, from position -772 to +40 (translation start ATG), included functional regulatory sequences capable of driving luciferase and EGFP expression, whereas shorter fragments from position -264 or -185 to +40 were insufficient to drive reporter activities.

Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes.

Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the "error prone" non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.

The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements.

BACKGROUND: Of the major families of long terminal repeat (LTR) retrotransposons, the Pao/BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni. RESULTS: A proviral form of Sinbad, a new LTR retrotransposon, was identified in the genome of S. mansoni. Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao/BEL-like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT), RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1, also from S. mansoni. It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans. Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca, Gulliver, Saci-2, Saci-3, and Fugitive, which are gypsy-like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs representing transcripts of Sinbad in numerous developmental stages of S. mansoni along with the identical 5'- and 3'-LTR sequences suggests that Sinbad is an active retrotransposon. CONCLUSION: Sinbad is a Pao/BEL type retrotransposon from the genome of S. mansoni. The Pao/BEL group appears to be comprised of at least five discrete subfamilies, which tend to cluster with host species phylogeny. Pao/BEL type elements appear to have colonized only the genomes of the Animalia. The distribution of these elements in the Ecdysozoa, Deuterostomia, and Lophotrochozoa is discontinuous, suggesting horizontal transmission and/or efficient elimination of Pao-like mobile genetic elements from some genomes.

Differential identification of Ascogregarina species (Apicomplexa: Lecudinidae) in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) by polymerase chain reaction.

We report 2 polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar gregarine species based on amplification of variable regions of the internal transcribed spacer region of ribosomal DNA. The gregarines we investigated were Ascogregarina barretti (Vavra), A. culicis (Ross), and A. taiwanensis (Lien and Levine), parasites of the mosquitoes Ochlerotatus triseriatus (Say), Aedes aegypti (Linnaeus), and Ae. albopictus (Skuse), respectively. These 3 important vector mosquitoes often utilize the same container habitats, where larval development and infection by the parasite occurs, leaving ample opportunity for cross-species gregarine infection. Because previous studies have shown that the parasites A. culicis and A. taiwanensis variably affect fitness in both normal and abnormal mosquito hosts, distinguishing parasite infection and species is important. The task is complicated by the fact that these 2 parasite species are virtually identical in morphology, whereas A. barretti is morphologically distinct. Of the 2 PCR-based assays reported here, the first provides a rapid, sensitive, and straight-forward means of general ascogregarine detection based on a single PCR amplification. The second method provides a means of differentiation between A. culicis and A. taiwanensis based on a species-specific PCR assay. Together, these assays allow whole mosquitoes to be tested for the presence of Ascogregarina species as well as identification of both A. culicis and A. taiwanensis singly or in dual infections.

Alu elements and DNA double-strand break repair.

Alu elements represent one of the most common sources of homology and homeology in the human genome. Homeologous recombination between Alu elements represents a major form of genetic instability leading to deletions and duplications. Although these types of events have been studied extensively through genomic sequencing to assess the impact of Alu elements on disease mutations and genome evolution, the overall abundance of Alu elements in the genome often makes it difficult to assess the relevance of the Alu elements to specific recombination events. We recently reported a powerful new reporter gene system that allows the assessment of various cis and trans factors on the contribution of Alu elements to various forms of genetic instability. This allowed a quantitative measurement of the influence of mismatches on Alu elements and instability. It also confirmed that homeologous Alu elements are able to stimulate non-homologous end joining events in their vicinity. This appears to be dependent on portions of the mismatch repair pathway. We are now in a position to begin to unravel the complex influences of Alu density, mismatch and location with alterations of DNA repair processes in various tissues and tumors.

Altering Genomic Integrity: Heavy Metal Exposure Promotes Transposable Element-Mediated Damage.

Maintenance of genomic integrity is critical for cellular homeostasis and survival. The active transposable elements (TEs) composed primarily of three mobile element lineages LINE-1, Alu, and SVA comprise approximately 30% of the mass of the human genome. For the past 2 decades, studies have shown that TEs significantly contribute to genetic instability and that TE-caused damages are associated with genetic diseases and cancer. Different environmental exposures, including several heavy metals, influence how TEs interact with its host genome increasing their negative impact. This mini-review provides some basic knowledge on TEs, their contribution to disease, and an overview of the current knowledge on how heavy metals influence TE-mediated damage.

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D.

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.