The Genetics of Dopa Decarboxylase in DROSOPHILA MELANOGASTER I. Isolation and Characterization of Deficiencies That Delete the Dopa-Decarboxylase-Dosage-Sensitive Region and the α-Methyl-Dopa-Hypersensitive Locus

A detailed cytogenetic investigation of 16 overlapping deficiencies in the 36C-40A region on the left arm of the second chromosome (2L) in Drosophila melanogaster is reported. These deficiencies permit a localization of both the dopa-decarboxylase-dosage-sensitive region and the α-methyl-dopa-hypersensitive locus, l(2)amd, to the same region, 37B10-37C7.     

Molecular Phylogeny Reveals the Past Transoceanic Voyages of Drywood Termites (Isoptera,Kalotermitidae)

Termites are major decomposers in terrestrial ecosystems and the second most diverse lineage of social insects. The Kalotermitidae form the second-largest termite family and are distributed across tropical and subtropical ecosystems, where they typically live in small colonies confined to single wood items inhabited by individuals with no foraging abilities. How the Kalotermitidae have acquired their global distribution patterns remains unresolved. Similarly, it is unclear whether foraging is ancestral to Kalotermitidae or was secondarily acquired in a few species. These questions can be addressed in a phylogenetic framework. We inferred time-calibrated phylogenetic trees of Kalotermitidae using mitochondrial genomes of ∼120 species, about 27% of kalotermitid diversity, including representatives of 21 of the 23 kalotermitid genera. Our mitochondrial genome phylogenetic trees were corroborated by phylogenies inferred from nuclear ultraconserved elements derived from a subset of 28 species. We found that extant kalotermitids shared a common ancestor 84 Ma (75–93 Ma 95% highest posterior density), indicating that a few disjunctions among early-diverging kalotermitid lineages may predate Gondwana breakup. However, most of the ∼40 disjunctions among biogeographic realms were dated at <50 Ma, indicating that transoceanic dispersals, and more recently human-mediated dispersals, have been the major drivers of the global distribution of Kalotermitidae. Our phylogeny also revealed that the capacity to forage is often found in early-diverging kalotermitid lineages, implying the ancestors of Kalotermitidae were able to forage among multiple wood pieces. Our phylogenetic estimates provide a platform for critical taxonomic revision and future comparative analyses of Kalotermitidae.     

A recombinant monoclonal-based Taenia antigen assay that reflects disease activity in extra-parenchymal neurocysticercosis

BackgroundAntigen tests for diagnosis and disease monitoring in some types of neurocysticercosis (NCC) are useful but access to testing has been limited by availability of proprietary reagents and/or kits.Methods/Principal findingsThree previously identified IgM-secreting hybridomas whose IgM products demonstrated specificity to Taenia solium underwent variable heavy and light chain sequencing and isotype conversion to mouse IgG. Screening of these recombinantly expressed IgG anti-Ts hybridomas, identified one (TsG10) with the highest affinity to crude Taenia antigen. TsG10 was then used as a capture antibody in a sandwich antigen detection immunoassay in combination with either a high titer polyclonal anti-Ts antibody or with biotinylated TsG10 (termed TsG10*bt). Using serum, plasma, and CSF samples from patients with active NCC and those from NCC-uninfected patients, ROC curve analyses demonstrated that the TsG10-TsG10-*bt assay achieved a 98% sensitivity and 100% specificity in detecting samples known to be antigen positive and outperformed the polyclonal based assay (sensitivity of 93% with 100% specificity). By comparing levels of Ts antigen (Ag) in paired CSF (n = 10) or plasma/serum (n = 19) samples from well-characterized patients with extra-parenchymal NCC early in infection and at the time of definitive cure, all but 2 (1 from CSF and 1 from plasma) became undetectable. There was a high degree of correlation (r = 0.98) between the Ag levels detected by this new assay and levels found by a commercial assay. Pilot studies indicate that this antigen can be detected in the urine of patients with active NCC.Conclusions/SignificanceA newly developed recombinant monoclonal antibody-based Ts Ag detection immunoassay is extremely sensitive in the detection of extra-parenchymal NCC and can be used to monitor the success of treatment in the CSF, serum/plasma and urine. The ability to produce recombinant TsG10 at scale should enable use of this antigen detection immunoassay wherever NCC is endemic.Clinical Trial RegistrationClinicalTrials.gov Identifiers: {"type":"clinical-trial","attrs":{"text":"NCT00001205","term_id":"NCT00001205"}}NCT00001205 - & {"type":"clinical-trial","attrs":{"text":"NCT00001645","term_id":"NCT00001645"}}NCT00001645.     

Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC-MALDI-MS/MS analysis

The isolation and analysis of glycoproteins by coupling lectin affinity chromatography with MS has emerged as a powerful strategy to study the glycoproteome of mammalian cells. However, this approach has not been used extensively for the analysis of plant glycoproteins. As with all eukaryotes, N-glycosylation is a common post-translational modification for plant proteins traveling through the secretory pathway. Many such proteins are destined for the cell wall, or apoplast, where they play important roles in processes such as modifying cell wall structure, sugar metabolism, signaling, and defense against pathogens. Here, we describe a strategy to enrich for and identify secreted plant proteins based on affinity chromatography using the lectin Concanavalin A and two-dimensional liquid chromatography, together with matrix-assisted laser desorption/ionization MS analysis. The value of this approach is illustrated through the characterization of glycoproteins that are expressed in ripe tomato (Solanum lycopersicum) fruit, a developmental stage that is fundamentally linked with significant changes in cell wall structure and composition. This glycoprotein trap strategy allowed the isolation of a sub-proteome with an extremely high proportion of proteins that are predicted to be resident in the cell wall or secretory pathway, and the identification of new putative cell wall proteins.     

Miocene to recent eolian dust record from the Southwest Pacific Ocean at 40° S latitude

A 14-meter long pelagic clay core recovered at Marlin Rise (40°00.531′S, 154°2.601′W; 4775 m water depth) in the Southwest Pacific Basin contains a record of eolian dust deposited since the early Miocene. Downcore analysis of detrital minerals reveals a dominantly eolian signature with relatively constant proportions of quartz, feldspar and illite and trace amounts of chlorite, kaolinite and smectite, consistent with a continental (loess-like) source region. Fish tooth Sr isotope stratigraphy reveals the base of the core to be 17.5 Ma, with low sedimentation rates (< 0.5 mm/kyr LSR) indicated for the interval 17.5 to 10 Ma; several hiatuses in deposition appear to be present upcore, but are beyond the age resolution of the fish teeth stratigraphy. These intervals are revealed as apparent discontinuities in the Sr isotope record, accompanied by pulses of anomalously rapid sedimentation at ~ 10 Ma, 6.7 Ma and 4.1 Ma. Bulk mass accumulation rates (MAR) are calculated at ~ 10 mg/cm²/kyr over the last 4 Myr, consistent with previously estimated Quaternary eolian flux rates to this part of the Pacific. Nd, Pb and Sr radiogenic isotopic compositions of the detrital mineral extract (< 38 µm) show no trends with age, while ⁴⁰Ar/³⁹Ar ages show an upcore younging trend (~180 Ma to ~150 Ma), in concert with a slight coarsening of eolian grain-size distributions. These ages likely reflect mixing of Mesozoic illite-dominated clay from at least two continental source areas: southeastern Australia (Murray–Darling Basin/Lake Eyre Basin) and New Zealand (South Island). The data indicate remarkable constancy of continental eolian sources exposed to weathering and dispersal at this latitude during the Neogene.     

The use of synthetic genes for the expression of ciliate proteins in heterologous systems

The common fish parasite, Ichthyophthirius multifiliis, expresses abundant glycosylated phosphatidylinositol (GPI)-anchored membrane proteins known as immobilization antigens, or i-antigens. These proteins are targets of the host immune response, and have been identified as potential candidates for recombinant subunit vaccine development. Nevertheless, because Ichthyophthirius utilizes a non-standard genetic code, expression of the corresponding gene products, either as subunit antigens in conventional protein expression systems, or as vector-encoded antigens in the case of DNA vaccines, is far from straightforward. To overcome this problem, we utilized 'assembly polymerase chain reaction' to manufacture synthetic versions of two genes (designated IAG52A[G5/CC] and IAG52B[G5/CC]) encoding approximately 52/55 kDa i-antigens from parasite strain G5. This approach made it possible to eliminate unwanted stop codons and substitute the preferred codon usage of channel catfish for the native sequences of the genes. To determine whether the synthetic alleles could be expressed in cells that use the standard genetic code, we introduced IAG52A[G5/CC] into a variety of heterologous cell types and tested for expression either by immunofluorescence light microscopy or Western blotting. When cloned downstream of appropriate promoters, IAG52A[G5/CC] was expressed in Escherichia coli, mammalian COS-7 cells, and channel catfish where it elicited antigen-specific immune responses. Interestingly, the localization pattern of the corresponding gene product in COS-7 cells indicated that while the protein was correctly folded, it was not present on the cell membrane, suggesting that the signal peptides required for GPI-anchor addition differ in ciliate and mammalian systems. Construction of synthetic alleles should have practical utility in the development of vaccines against Ichthyophthirius, and at the same time, provide a general method for the expression of ciliate genes in heterologous systems.     

Pro-Thyrotropin-Releasing Hormone Processing by Recombinant PC1

Abstract: Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH₂), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [³H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH_25–152 or preproTRH_25–158) and a 10-kDa C-terminal peptide (preproTRH_154–255 or preproTRH_160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (preproTRH_25–76 or preproTRH_25–82) and a 3.8-kDa peptide (preproTRH_83–108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (preproTRH_206–255). The optimal pH for these cleavages was 5.5. ZnCl₂, EDTA, EGTA, and the omission of Ca²⁺ inhibited the formation of pYE₂₇ (preproTRH_25–50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.     

Fusing structure and function: a structural view of the herpesvirus entry machinery

Herpesviruses are double-stranded DNA, enveloped viruses that infect host cells through fusion with either the host cell plasma membrane or endocytic vesicle membranes. Efficient infection of host cells by herpesviruses is remarkably more complex than infection by other viruses, as it requires the concerted effort of multiple glycoproteins and involves multiple host receptors. The structures of the major viral glycoproteins and a number of host receptors involved in the entry of the prototypical herpesviruses, the herpes simplex viruses (HSVs) and Epstein-Barr virus (EBV), are now known. These structural studies have accelerated our understanding of HSV and EBV binding and fusion by revealing the conformational changes that occur on virus-receptor binding, depicting potential sites of functional protein and lipid interactions, and identifying the probable viral fusogen.     

Net‐spinning caddisfly distribution in large regulated rivers

1. Most of the world's large rivers are dammed for the purposes of water storage, flood control, and power production. Damming rivers fundamentally alters water temperature and flows in tailwater ecosystems, which in turn affects the presence and abundance of downstream biota.
2. We collaborated with more than 200 citizen scientists to collect 2,194 light trap samples across 2 years and more than 2,000 river km. Samples contained 16,222 net‐spinning caddisfly (Hydropsyche) individuals across six species. We used these data to model the distribution of Hydropsyche throughout the Colorado River Basin in the western U.S.A. to identify the roles of water temperature, flows, and species‐specific morphology in determining aquatic species distributions throughout a large arid watershed that has been heavily altered by damming.
3. We predicted that water temperatures would determine Hydropsyche presence and abundance to a greater extent than diel variation in river stage associated with hydropower production. Among many species, adult female Hydropsychids are morphologically adapted to swim to deep‐water oviposition sites. We predicted that the presence of this ability would negate the otherwise deleterious effects of high stage change on caddisfly egg mortality.
4. We found that distributions of the two most widespread species, Hydropsyche occidentalis and Hydropsyche oslari (92% of total Hydropsyche captured), were both predicted by water temperatures. However, we also found that the abundance of H. oslari decreased by as much as 10‐fold as diel stage change increased, despite the presence of female morphological adaptations for deep‐water oviposition. We found sexual dimorphism and evidence for deep‐water swimming adaptations in 5/6 species.
5. Our results show that net‐spinning caddisflies have species‐specific responses to environmental variation and suggest that environmental flows designed to reduce diel stage change and destabilise water temperatures may improve habitat quality for these ubiquitous and important aquatic insects.