Transgenesis in Strongyloides and related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing.

Transgenesis for Strongyloides and Parastrongyloides was accomplished in 2006 and is based on techniques derived for Caenorhabditis elegans over two decades earlier. Adaptation of these techniques has been possible because Strongyloides and related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. In Strongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes in S. stercoralis requires their integration into the genome, and stable lines have been derived from integrants created using the piggyBac transposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis in Strongyloides and Parastrongyloides opens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.

Effect of short-term heat acclimation with permissive dehydration on thermoregulation and temperate exercise performance.

We examined the effect of short-term heat acclimation with permissive dehydration (STHADe) on heat acclimation (HA) and cycling performance in a temperate environment. Ten trained male cyclists [mean (SD) maximal oxygen uptake: 63.3(4.0) mL/kg/min; peak power output (PPO): 385(40) W; training: 10 (3) h/week] underwent a STHADe program consisting of 5 days of exercise (maximum 90 min/day) in a hot environment (40 °C, 50% RH) to elicit isothermic heat strain [rectal temperature 38.64(0.27) °C]. Participants abstained from fluids during, and 30 min after, HA sessions. Pre- and post-STHADe HA was evaluated during euhydrated fixed-intensity exercise (60 min) in hot conditions; the effect of STHADe on thermoregulation was also examined under temperate conditions (20 min fixed-intensity exercise; 22 °C, 60% RH). Temperate cycling performance was assessed by a graded exercise test (GXT) and 20-km time trial (TT). STHADe reduced thermal and cardiovascular strain in hot and temperate environments. Lactate threshold [Δ = 16 (17) W] and GXT PPO [Δ = 6 (7) W] were improved following STHADe (P < 0.05), but TT performance was not affected (P > 0.05), although there was a trend for a higher mean power (P = 0.06). In conclusion, STHADE can reduce thermal and cardiovascular strain under hot and temperate conditions and there is some evidence of ergogenic potential for temperate exercise, but longer HA regimens may be necessary for this to meaningfully influence performance.

PCR amplification of putative gpa-2 and gpa-3 orthologs from the (A+T)-rich genome of Strongyloides stercoralis.

Two G protein alpha subunit genes orthologous to gpa-2 and gpa-3 in Caenorhabditis elegans have been identified in the parasitic nematode, Strongyloides stercoralis. These genes mediate chemosensory signal transduction regulating dauer arrest in C. elegans. In the parasite, they represent candidate mediators for regulation of the choice between free-living and parasitic life cycles, the obligatory developmental arrest of infective larvae, and reactivation of development after infection. The (A+T) content of these genes is 72.2% for coding sequences, 90% for introns, and 84.1% for 5' and 3' flanking regions, requiring the use of low extension temperatures for long distance PCR. The possible significance of conserved structural motifs of these proteins is discussed.

The evolution of hexamerins and the phylogeny of insects.

The evolutionary relationships among arthropod hemocyanins and insect hexamerins were investigated. A multiple sequence alignment of 12 hemocyanin and 31 hexamerin subunits was constructed and used for studying sequence conservation and protein phylogeny. Although hexamerins and hemocyanins belong to a highly divergent protein superfamily and only 18 amino acid positions are identical in all the sequences, the core structures of the three protein domains are well conserved. Under the assumption of maximum parsimony, a phylogenetic tree was obtained that matches perfectly the assumed phylogeny of the insect orders. An interesting common clade of the hymenopteran and coleopteran hexamerins was observed. In most insect orders, several paralogous hexamerin subclasses were identified that diversified after the splitting of the major insect orders. The dipteran arylphorin/LSP-1-like hexamerins were subject to closer examination, demonstrating hexamerin gene amplification and gene loss in the brachyceran Diptera. The hexamerin receptors, which belong to the hexamerin/hemocyanin superfamily, diverged early in insect evolution, before the radiation of the winged insects. After the elimination of some rapidly or slowly evolving sequences, a linearized phylogenetic tree of the hexamerins was constructed under the assumption of a molecular clock. The inferred time scale of hexamerin evolution, which dates back to the Carboniferous, agrees with the available paleontological data and reveals some previously unknown divergence times among and within the insect orders.

The Drosophila Lsp-1 beta gene. A structural and phylogenetic analysis.

In Drosophila melanogaster, metamorphosis and reproduction are thought to be supported in large by two immunologically distinct hexameric storage proteins (hexamerins), larval serum protein 1 (LSP-1), a mixed hexamer of three closely related subunits, Lsp-1 (alpha, beta and gamma) and larval serum protein 2 (LSP-2), a homohexamer of Lsp-2 subunits. To understand the structural and functional differences between these two storage hexamers, the nucleotide sequence of the coding region of the Lsp-1 beta gene was determined for comparison with LSP-2 and a number of other arthropod hexamerins. The G + C content of the coding sequence is 55%, with 92.8% of the codons containing G or C in the third position. Conceptual translation of the Lsp-1 beta open reading frame revealed a 789-amino-acid polypeptide of 94465 Da. The amino acid sequence of Lsp-1 beta is 65.8% identical to that of calliphorin, the major hexamerin of the blowfly, Calliphora vicina, and only 35.2% identical to Drosophila Lsp-2. This greater similarity to calliphorin is also reflected in high aromatic amino acid and methionine contents, in contrast to LSP-2 which is enriched to a lesser extent only in aromatic amino acids. Lsp-1 beta is also more closely related to calliphorin with respect to the protein domain structure, the presence of a single intron in its gene, and the absence of glycosylation sites. However, phylogenetic analysis based on multiple alignments revealed that LSP-1 calliphorin and LSP-2 form a distinct dipteran clade whose members are more similar to each other than to any previously sequenced lepidopteran hexamerin or arthropod hemocyanin.

Properties and significance of a riboflavin-binding hexamerin in the hemolymph of Hyalophora cecropia.

A riboflavin-binding hexamerin isolated from pupal hemolymph of Hyalophora cecropia has a native M(r) of 510,000, subunit M(r) of 85,000, and a 5% carbohydrate content. An intrachain cross-link was confirmed in protease limit digests. Ellman titration confirmed the presence of a sulfhydryl group, which is needed for this linkage. Though Cu2+ is known to promote the linkage, heavy metals were not detected in the isolate. Heat denaturation released ligand with the absorbency, fluorescence spectra, and chromatographic behavior of riboflavin. Binding resulted in substantial quenching of the fluorescence of both the isoalloxazine in riboflavin and of aromatic groups in the apoprotein. Kinetic analysis indicated a KD of 2.5 x 10(-7) M for riboflavin, 1.3 x 10(-7) M for lumiflavin, and greater than 1 x 10(-6) M for FMN and FAD. Over four moles of flavin were bound per mole of hexamerin. The amount of riboflavin in pupal hemolymph is sufficient to occupy only 2-3 of these sites. Riboflavin is also associated with lipophorin and vitellogenin, but the molar ratios after protein isolation were low. On a standard laboratory diet, riboflavin is in great excess, but most of it is apparently excreted before the apoprotein first appears in the hemolymph, just before wandering. The concentration of riboflavin-binding hexamerin rises to 15-30 mg/ml in pupae; relative to other hexamerins, very little is stored in the fat body. All of the apoprotein and 75% of riboflavin disappear from the hemolymph during adult development. An amount of flavin at least equal to that stored in pupal hemolymph is transferred to the eggs formed during this period.

Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells.

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield.