Vector competence of Coquillettidia linealis (Skuse) (Diptera: Culicidae) for Ross River and Barmah Forest viruses

Abstract Coquillettidia linealis is a severe pest on some of the Moreton Bay islands in Queensland, Australia, but little is known of its breeding habitats and biology. Because of its high abundance and its association with Ross River (RR) and Barmah Forest (BF) viruses by field isolation, its vector competence was evaluated in the laboratory by feeding dilutions of both viruses in blood. For RR, Cq. linealis was of comparable efficiency to Ochlerotatus vigilax (Skuse), recognised as being a major vector. Results were as follows for Cq. linealis and Oc. vigilax , respectively: dose to infect 50%, 10^2.2 and <10^1.7 CCID₅₀/mosquito; 88% and 90% disseminated infection at 4 days postinfection; transmission at 4 days with rates of 68−92% and 25−60%. For BF dose to infect 50%, 10^2.7 and 10^2.0; disseminated infection rates on first transmission day (day 6), 40% and 70%; transmission rates of 8−16% and 0−10%. As a capillary-tube method was used rather than suckling mice to demonstrate transmission, transmission rates may be underestimates. This, the first study of the vector competence of Cq. linealis in Australia, demonstrates that this species deserves control on the southern Moreton Bay islands.     

Size scaling of whole‐body metabolic activity in the noble crayfish (Astacus astacus) estimated from measurements on a single leg

1. Use of electron transport system (ETS) activity in a single leg for estimating whole‐body ETS activity was explored in the noble crayfish Astacus astacus. Oxygen consumption and ETS activity of the whole body and of a walking leg were measured in different‐sized animals at 10 °C to compare the size scaling of oxygen consumption, whole‐body ETS activity and the ratio of whole‐body ETS activity to oxygen consumption (ETS/R). 2. Electron transport system activity of a leg and the ratio of ETS activity of a whole crayfish to that of a leg were correlated with wet mass of animals. Therefore, metabolic potential in whole noble crayfish can be estimated on the basis of the measured ETS activity in a single leg and crayfish mass. This approach provides a valuable tool for determining metabolic characteristics of crayfish without killing them. 3. Mass‐specific oxygen consumption decreased with increasing wet mass, while ETS activity of whole crayfish showed no significant correlation with wet mass. Both oxygen consumption and ETS activity correlated significantly with protein mass. 4. The increase in ETS/R with increasing wet mass of the noble crayfish indicates that small organisms exploit a greater proportion of their metabolic potential for standard metabolism than larger ones. This is the first report on ETS/R in crayfish.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

A new cardiid bivalve from the Pliocene Baklan Basin (Turkey) and the origin of modern Ponto‐Caspian taxa

Abstract: We present the first record of the cardiid genus Monodacna from the Pliocene of Anatolia, Turkey. Monodacna imrei sp. nov. was found in the Pliocene Killik Formation from the western margin of the Baklan Basin, in very marginal brackish to freshwater lacustrine deposits. The new record extends the stratigraphic range of the modern Ponto‐Caspian genus back into the Pliocene and adds to earlier evidence that modern Ponto‐Caspian taxa originated in the Pliocene of south‐western Turkey.     

Ionization and solubility of chitosan solutions related to thermosensitive chitosan/glycerol-phosphate systems

Chitosan is a linear cationic biopolymer composed of glucosamine and N-acetyl-glucosamine that is only soluble in acidic aqueous solutions and precipitates when neutralized. However, it was recently discovered that chitosan dissolved in solutions containing glycerol phosphate was soluble at near neutral pH and produced a sol-gel transition when heated. Understanding this unique thermogelling system requires improved characterization of the ionization and solubility behaviors of chitosan, in particular dependencies on temperature, salt, chitosan concentration, and fD, where fD is the fraction of glucosamine monomers (deacetylated monomers) in chitosan. In the current study we performed temperature-controlled titration and dilution experiments on chitosan solutions with fD of 0.72, 0.85, and 0.98 at concentrations ranging from 1.875 to 30 mM of its glucosamine monomer and with 0 to 150 mM added salt. Light transmittance measurements were performed during titration to indicate precipitation. We found the apparent proton dissociation constant of chitosan, pKap, to (1) decrease strongly with increased temperature, (2) increase strongly with increased salt, (3) increase strongly with increased chitosan concentration in low-salt conditions, and (4) decrease weakly with increasing fD. All of the above influences on chitosan pKap were accurately predicted using a mean-field Poisson-Boltzmann (PB) cylindrical cell model where the only adjustable parameter was the temperature-dependent chitosan intrinsic monomeric dissociation constant pK0(T). The resulting chitosan pK0 values at 25 degrees C were in the range from 6.63 to 6.78 for all chitosans and salt contents tested. The temperature dependence of chitosan ionization was found to strongly reduce pK0(T) by 0.023 units per degrees C, for example, resulting in a reduction of chitosan pK0(T) from 7.1 at 5 degrees C to 6.35 at 37 degrees C for fD of 0.72 in 150 mM salt. A similar temperature-dependent reduction of the pKa of the glucosamine monomer was found (-0.027 units per degrees C) while the pKa of glycerol phosphate did not change significantly with temperature. The latter result suggested that chitosan solutions heated in the presence of glycerol phosphate will become partly neutralized by transferring protons to glycerol phosphate and thereby allow attractive interchain forces to form a physically cross-linked gel under the appropriate conditions. Additionally, the degree of ionization of chitosan when it precipitates upon addition of a strong base was measured to be in the range from 0.25 to 0.55 and was found to (1) be insensitive to temperature, (2) increase strongly with increased salt, and (3) increase strongly with fD. The salt effect was accounted for by the PB model, while the influence of fD appeared to be due to acetyl groups impeding attractive chain-to-chain association to increase solubility and require reduced ionization levels to precipitate.     

Description of Paratetrahymena parawassi n. sp. using Morphological and Molecular Evidence and a Phylogenetic Analysis of Paratetrahymena and Other Taxonomically Ambiguous Genera in the Order Loxocephalida (Ciliophora,Oligohymenophorea)

ABSTRACT. The marine scuticociliate Paratetrahymena parawassi n. sp. is described on the basis of morphology, especially infraciliature, and the sequence of its small subunit (SSU) rRNA gene to become the second known member of its genus. Paratetrahymena and other ciliates in the order Loxocephalida possess a mixture of morphological and morphogenetic features characteristic of the subclasses Hymenostomatia and Scuticociliatia. Accordingly, we used SSU rRNA sequences to analyze the phylogeny of Paratetrahymena and three other loxocephalid genera. Paratetrahymena and Cardiostomatella vermiformis formed a moderately well‐supported clade that diverged at a deep level from all other scuticociliates, supporting separation of loxocephalids from other scuticociliates as a suprafamilial taxon. Sathrophilus holtae was a sister taxon to Paratetrahymena and Cardiostomatella in a poorly supported, unresolved relationship; nevertheless, association of all three genera into a single clade was supported by an approximately unbiased (AU) test. Any association of these genera singly or as a group with the Hymenostomatia was rejected decisively by AU tests and by a complete absence in the loxocephalids of the unique nucleotide identities that distinguish hymenostomes. Therefore, the morphological and morphogenetic similarities of loxocephalids to hymenostomes may be plesiomorphies, and the conflicting mix of scuticociliate and hymenostome characteristics seen in loxocephalids may result from differing rates of character evolution. Dexiotrichides pangi and Urocentrum, which is currently classified as a peniculid, formed a small clade that associated with hymenostomes and peritrichs. Monophyly of the Loxocephalida with Dexiotrichides and/or Urocentrum included was not rejected by AU; however, inclusion of Urocentrum in the Peniculia was rejected by AU tests. A hypothesis is offered to explain the lack of resolution of loxocephalid ciliates and Urocentrum in phylogenetic trees, namely that their phylogenetic positions are influenced by a combination of heterogeneous data and long‐branch attraction caused by poor representation of taxa in analyses. The well‐known genus Cyclidium, a member of the order Pleuronematida, was revealed to be polyphyletic as a byproduct of our analyses of loxocephalids. In particular, Cyclidium porcatum appears to fall outside the clade containing typical members of the subclass Scuticociliatia and thus invites investigation as a possible member of the order Loxocephalida.     

Increased apoptosis in cryopreserved autologous hematopoietic progenitor cells collected by apheresis and delayed neutrophil recovery after transplantation: a nested case-control study

Background aimsDelayed neutrophil recovery following autologous hematopoietic stem cell transplantation (aHSCT) increases transplant-related morbidity. Apoptosis induced by cryopreservation and thawing of hematopoietic progenitor cells collected by apheresis (HPC-A) was investigated in this nested case-control study as a factor associated with delayed neutrophil recovery following aHSCT.MethodsAmong patients with lymphoma who underwent aHSCT between 2000 and 2007 (n = 326), 13 cases of primary delayed neutrophil recovery and 22 age- and sex-matched controls were identified. Apoptosis and viability were measured using multiparameter flow cytometry, and colony-forming capacity was determined using semi-solid methylcellulose assays.ResultsHPC-A grafts from cases and controls had similar percentages of viable mononuclear cells (MNC) and CD34⁺progenitor cells, as determined by standard 7AAD dye exclusion methods measured before and after cryopreservation. Patients with delayed neutrophil recovery received increased numbers of apoptotic MNC (P = 0.02) but similar numbers of apoptotic CD34⁺ cells per kilogram measured after thawing. Apoptosis was more pronounced in MNC compared with CD34⁺ cells after thawing, and apoptosis was negligible in freshly collected HPC-A products. Patients with delayed neutrophil recovery had fewer total colony-forming unites (CFU) and CFU-granulocyte–macrophages (GM) per 10⁵ viable post-thaw MNC compared with controls (P < 0.05).ConclusionsIncreased numbers of apoptotic MNC in thawed HPC-A products are associated with delayed neutrophil recovery after aHSCT. Studies that address factors contributing to increased apoptosis are needed, and measuring apoptosis in thawed HPC-A may have a role in the assessment of graft adequacy.     

A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification

Characterization of regions flanking a known sequence within a genome, known as genome walking, is a cornerstone technique in modern genetic analysis. In the present work we have developed a new PCR-dependent, directional genome walking protocol based on the unique circularization property of a novel DNA ligase, CircLigase. In the first step, PCR based primer extension is performed using a phosphorylated primer, designed to extend from the boundary of the known sequence, into the flanking region. This linear amplification results in the generation of single-stranded (ss) DNA, which is then circularized using CircLigase. Using the hyperbranching activity of Phi29 DNA polymerase, the circular ssDNA is then linearized by rolling circle amplification, resulting in copious amounts of double stranded concatameric DNA. Nested primers are used to amplify the flanking sequence using inverse PCR. The products are resolved on an agarose gel and the bands whose mobility change due to the nested location of the primer combination used are identified, extracted, and cloned into a plasmid vector for sequencing. Empirical proof for this concept was generated on two antimicrobial biosynthetic genes in Pseudomonas sp. LBUM300. Using the hcnB and phlD genes as starting points, ca 1 kb of flanking sequences were successfully isolated. The use of locus specific primers ensured both directionality and specificity of the walks, alleviating the generation of spurious amplicons, typically observed in randomly primed walking protocols. The presented genome walking protocol could be applied to any microbial genome and requires only 100-150 bp of prior sequence information. The proposed methodology does not entail laborious testing of restriction enzymes or adaptor ligation. This is the first report of a successful application of the novel ligase enzyme, CircLigase for genomic walking purposes.