First report of metazoan parasites from the cichlid Pseudocrenilabrus philander and the cyprinid Enteromius paludinosus in a South African Ramsar wetland

Parasites of two small fish species from a Ramsar wetland in South Africa were studied in 2014–2015. The cichlid Pseudocrenilabrus philander (Weber, 1897) was parasitised by the copepod Lernaea cyprinacea Linnaeus, 1758, the monogenean Gyrodactylus thlapi Christison, Shinn & van As, 2005 and four gryporhynchid metacestode (Cyclophyllidea) species: Paradilepis scolecina (Rudolph, 1819), Paradilepis maleki Khalil, 1961, Neogryporhynchus lasiopeius Baer & Bona, 1960 and Valipora campylancristrota (Wedl, 1855). The cyprinid Enteromius paludinosus (syn. Barbus paludinosus) (Peters, 1852) was infected with the monogenean parasites Dogielius intorquens Crafford, Luus-Powell & Avenant-Oldewage, 2012, Dactylogyrus teresae Mashego, 1983, and three Dactylogyrus spp. These results represent several new locality as well as host records and further contribute information on the parasitic diversity in the Barberspan Ramsar wetland.     

Expression and purification of recombinant human fibroblast growth factor receptor in Escherichia coli

Human fibroblast growth factor receptor (FGFR) is responsible for multifunctional signaling that regulates developmental processes. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) include the determinants of ligand binding and specificity for fibroblast growth factor and heparan sulfate. D1 and the D1-D2 linker with a contiguous stretch of acidic amino acids are known to be involved in auto-inhibitory regulation. In an effort to gain a better understanding of the role of D1 and the linker in FGFR regulation, we have subcloned, overexpressed, and purified the extracellular fragments, D1-D2 and D1-D3, of FGFR1 in Escherichia coli. The recombinant proteins were produced in an insoluble form and were renatured using a dropwise or on-column refolding method. In addition, D2-D3 was coexpressed with chaperones to test the possibility that the presence of chaperones might enhance refolding efficiencies. A combination of immobilized nickel and heparin affinity chromatography and size-exclusion chromatography resulted in the purification of recombinant ectodomain proteins D1-D2 and D1-D3 of high purity for structural studies.     

Remating inhibition in female Queensland fruit flies: effects and correlates of sperm storage

Reproductive success of male insects commonly hinges both on their ability to secure copulations with many mates and also on their ability to inseminate and inhibit subsequent sexual receptivity of their mates to rival males. We here present the first investigation of sperm storage in Queensland fruit flies (Tephritidae: Bactrocera tryoni; a.k.a. 'Q-flies') and address the question of whether remating inhibition in females is directly influenced by or correlated with number of sperm stored from their first mates. We used irradiation to disrupt spermatogenesis and thereby experimentally reduce the number of sperm stored by some male's mates while leaving other aspects of male sexual performance (mating probability, latency until copulating, copula duration) unaffected. Females that mated with irradiated rather than normal males were less likely to store any sperm at all (50% vs. 89%) and, if some sperm were stored, the number was greatly reduced (median 11 vs. 120). Despite the considerable differences in sperm storage, females mated by normal males and irradiated males were similarly likely to remate at the next opportunity, indicating (1) number of sperm stored does not directly drive female remating inhibition and (2) factors actually responsible for remating inhibition are similarly expressed in normal and irradiated males. While overall levels of remating were similar for mates of normal and irradiated males, factors responsible for female remating inhibition were positively associated with presence and number of sperm stored by mates of normal but not irradiated males. We suggest seminal fluids as the most likely factor responsible for remating inhibition in female Q-flies, as these are likely to be transported in proportion to number of sperm in normal males, be uninfluenced by irradiation, and be transported without systematic relation to sperm number in irradiated males.     

Glycosylation of DsbA in Francisella tularensis subsp. tularensis

In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis.     

A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD

The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal-dominant genes underlying these common neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK, and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one-third of familial ALS cases of outbred European descent, making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.     

Network quantitative trait loci mapping of circadian clock outputs identifies metabolic pathway-to-clock linkages in Arabidopsis

Modern systems biology permits the study of complex networks, such as circadian clocks, and the use of complex methodologies, such as quantitative genetics. However, it is difficult to combine these approaches due to factorial expansion in experiments when networks are examined using complex methods. We developed a genomic quantitative genetic approach to overcome this problem, allowing us to examine the function(s) of the plant circadian clock in different populations derived from natural accessions. Using existing microarray data, we defined 24 circadian time phase groups (i.e., groups of genes with peak phases of expression at particular times of day). These groups were used to examine natural variation in circadian clock function using existing single time point microarray experiments from a recombinant inbred line population. We identified naturally variable loci that altered circadian clock outputs and linked these circadian quantitative trait loci to preexisting metabolomics quantitative trait loci, thereby identifying possible links between clock function and metabolism. Using single-gene isogenic lines, we found that circadian clock output was altered by natural variation in Arabidopsis thaliana secondary metabolism. Specifically, genetic manipulation of a secondary metabolic enzyme led to altered free-running rhythms. This represents a unique and valuable approach to the study of complex networks using quantitative genetics.     

High plasma thiocyanate levels modulate protein damage induced by myeloperoxidase and perturb measurement of 3-chlorotyrosine

Smokers have an elevated risk of atherosclerosis but the origin of this elevated risk is incompletely defined, though increasing evidence supports a role for the oxidant-generating enzyme myeloperoxidase (MPO). In previous studies we have demonstrated that smokers have elevated levels of thiocyanate ions (SCN(-)), relative to nonsmokers, and increased thiol oxidation, as SCN(-) is a favored substrate for MPO, and the resulting hypothiocyanous acid (HOSCN) targets thiol groups rapidly and selectively. In this study we show that increased HOSCN formation by MPO diminishes damage to nonthiol targets on both model proteins and human plasma proteins. Thus high SCN(-) levels protect against HOCl- and MPO-mediated damage to methionine, tryptophan, lysine, histidine, and tyrosine residues on proteins. Furthermore, levels of the HOCl-mediated marker compound 3-chlorotyrosine and the cross-linked product dityrosine are decreased. Plasma protein 3-chlorotyrosine levels induced by HOCl exposure in nonsmokers are elevated over the levels detected in smokers when exposed to identical oxidative insult (P<0.05), and a strong inverse correlation exists between plasma SCN(-) levels and 3-chlorotyrosine concentrations (r=0.6182; P<0.0001). These correlations were also significant for smokers (r=0.2724; P<0.05) and nonsmokers (r=0.4141; P<0.01) when analyzed as individual groups. These data indicate that plasma SCN(-) levels are a key determinant of the extent and type of protein oxidation induced by MPO on isolated and plasma proteins and that smoking status and resulting high SCN(-) levels can markedly modulate the levels of the widely used biomarker compound 3-chlorotyrosine.