HiTRACE: high-throughput robust analysis for capillary electrophoresis

MOTIVATION: Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment. RESULTS: We propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. We illustrate the application of HiTRACE on 13 datasets representing 4 different RNAs, 3 chemical modification strategies and up to 480 single mutant variants; the largest datasets each include 87 360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate datasets. Furthermore, while the smallest of these datasets required 7-10 h of manual intervention using prior approaches, HiTRACE quantitation of even the largest datasets herein was achieved in 3-12 min. The HiTRACE method, therefore, resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands.     

Repellent plants provide affordable natural screening to prevent mosquito house entry in tropical rural settings--results from a pilot efficacy study

Sustained malaria control is underway using a combination of vector control, prompt diagnosis and treatment of malaria cases. Progress is excellent, but for long-term control, low-cost, sustainable tools that supplement existing control programs are needed. Conventional vector control tools such as indoor residual spraying and house screening are highly effective, but difficult to deliver in rural areas. Therefore, an additional means of reducing mosquito house entry was evaluated: the screening of mosquito house entry points by planting the tall and densely foliated repellent plant Lantana camara L. around houses. A pilot efficacy study was performed in Kagera Region, Tanzania in an area of high seasonal malaria transmission, where consenting families within the study village planted L. camara (Lantana) around their homes and were responsible for maintaining the plants. Questionnaire data on house design, socioeconomic status, malaria prevention knowledge, attitude and practices was collected from 231 houses with Lantana planted around them 90 houses without repellent plants. Mosquitoes were collected using CDC Light Traps between September 2008 and July 2009. Data were analysed with generalised negative binomial regression, controlling for the effect of sampling period. Indoor catches of mosquitoes in houses with Lantana were compared using the Incidence Rate Ratio (IRR) relative to houses without plants in an adjusted analysis. There were 56% fewer Anopheles gambiae s.s. (IRR 0.44, 95% CI 0.28-0.68, p<0.0001); 83% fewer Anopheles funestus s.s. (IRR 0.17, 95% CI 0.09-0.32, p<0.0001), and 50% fewer mosquitoes of any kind (IRR 0.50, 95% CI 0.38-0.67, p<0.0001) in houses with Lantana relative to controls. House screening using Lantana reduced indoor densities of malaria vectors and nuisance mosquitoes with broad community acceptance. Providing sufficient plants for one home costs US $1.50 including maintenance and labour costs, (30 cents per person). L. camara mode of action and suitability for mosquito control is discussed.     

Failure of CD4 T-cells to respond to liver-derived antigen and to provide help to CD8 T-cells

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.     

Ultrastructural Observations on Fertilization in Dionchus remorae (Platyhelminthes,Monogenea, Dionchidae)

Ultrastructural observations are presented for some of the stages occurring during fertilization in Dionchus remorae (a gill parasite of Echeneis naucrates) and are believed to be the first published concerning a monogenean. Fertilized female germ cells were found in the ovary. Several loops of the spermatozoon were present within the oocyte cytoplasm; the sperm nucleus became electron lucent and the parallel peripheral doublets of the axonemes became increasingly divergent. The cortical granules in the oocyte were not released immediately after penetration by the spermatozoon. The homogeneity apparently found in the oocyte ultrastructure and process of fertilization in the monogeneans and digeneans contrasts with the variety that exists in their sperm ultrastructure.     

Screening for bacterial-fungal associations in a south-eastern US salt marsh using pre-established fungal monocultures

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated community. We examined whether physical associations exist between individual bacterial and fungal species that co-occur on decaying smooth cordgrass, Spartina alterniflora, in a south-eastern US salt marsh. Fungal-pervaded decaying Spartina was used as "bait" for potential bacterial associates. The bundles (infiltrated with one of three dominant fungal members of the decomposer assemblage, or an autoclaved control) were placed in a salt marsh and collected biweekly for 6 weeks during the first experiment (late summer 2002), and weekly for 3 weeks during the second experiment (early summer 2003). Terminal-restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to track colonization by bacterial taxa in association with the established fungal species. T-RFLP analysis of 18S-to-28S internal transcribed spacer (ITS) regions was used to monitor changes in fungal communities once bundles had been placed in the field. Results from both years were nearly identical, and showed that invasion by fungi other than the bait species was slow, resulting in a virtual fungal monoculture for several weeks into the experiments. Surprisingly, bacterial communities were unaffected by the identity of the fungal bait. Regardless of the fungal species, and even in the absence of prior fungal colonization, bacterial 16S rRNA profiles were remarkably similar. These results suggest that few species-specific associations, either positive or negative, exist between bacterial and fungal members of the Spartina decomposer community during initial colonization.     

Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of measurements of specimens prepared using different methods, and a description of P. caledonicus n. sp.

Species of Pseudorhabdosynochus were studied from fresh specimens collected from Epinephelus fasciatus and E. merra off New Caledonia, South Pacific, and specimens deposited in Museums. Experiments on two species demonstrated that the sclerotised hollow organs, such as the quadriloculate male copulatory organ and the vagina, may show differences in measurements of up to 50% when flattened. P. caledonicus n. sp. is described from E. fasciatus in New Caledonia, on which it is relatively rare; it is distinguished on the basis of the quadriloculate organ, which has a very thin anterior wall, the sclerotised parts of the vagina in form of a straight tube with a star-shaped lateral structure, and the squamodiscs composed of 11 open rows of rodlets. P. cupatus (Young, 1969) is redescribed from abundant material from E. fasciatus off New Caledonia (new geographical record) and compared with paratype specimens from Australia (from E. fasciatus and E. merra) and specimens from E. fasciatus in the Red Sea (both herein redescribed and figured); a specimen was also found on a slide from E. merra off Vanuatu. P. melanesiensis (Laird, 1958) is redescribed from material from E. merra off New Caledonia (new geographical record) and compared with type-specimens (herein redescribed and figured) from the same host off Vanuatu. The structure of the sclerotised vagina in P. cupatus and P. melanesiensis is very similar, with a thin-walled tube and a heavily sclerotised structure with three loculi. P. vagampullum (Young, 1969) is redescribed from the paratypes from E. merra from Australia, but was not found in New Caledonia; specimens included among its paratypes (from E. merra in Australia), but different, are herein attributed to Pseudorhabdosynochus sp. 3. P. lantauensis (Beverley-Burton & Suriano, 1981) is redescribed from the paratype specimens from E. longispinis off Hong-Kong. A specimen found among the paratypes of P. cupatus belongs to a different species, herein designated as Pseudorhabdosynochus sp. 1. Specimens from E. longispinis off Hong-Kong, previously attributed to P. cupatus, are attributed to another species, Pseudorhabdosynochus sp. 2. The three species P. cupatus, Pseudorhabdosynochus sp. 1 and Pseudorhabdosynochus sp. 2 have in common a 'lamellosquamodisc' composed of central telescopic lamellae and peripheral rows of rodlets; they can be distinguished by the shape of the sclerotised vagina and measurements of the haptoral hard-parts. Specimens from E. longispinis off Hong-Kong, previously attributed to P. vagampullum, probably belong to a different species. Consequently, after these modified determinations, P. cupatus parasitises only E. fasciatus and E. merra, and P. melanesiensis and P. vagampullum parasitise only E. merra. With their wide geographical distribution and different species of Pseudorhabdosynochus in different localities, E. fasciatus and E. merra appear to represent excellent models for investigating monogenean biogeography in the Indo-Pacific Ocean.     

Two anisakid nematodes from marine fishes off New Caledonia, including Raphidascaris (Ichthyascaris) nemipteri n. sp. from Nemipterus furcosus

One new and one known species of the ascaridoid family Anisakidae are reported from marine fishes off the southwestern coast of New Caledonia: Raphidascaris (Ichthyascaris) nemipteri n. sp. from the intestine of the forked-tailed threadfin bream Nemipterus furcosus (Nemipteridae, Perciformes) and Hysterothylacium cenaticum (Bruce & Cannon, 1989) from the intestine of the striped marlin Tetrapturus audax (Istiophoridae, Perciformes). R. nemipteri is characterised mainly by the shape (wider than long) of the lips, the length of the spicules (225–399 μm, which represent 2.7–4.2% of the body length), the number (22–33) of caudal pre-anal papillae, the position of the vulva (at 16–20% of the body length), and the presence of cuticular spines on the tip of the female tail. Specimens of H. cenaticum from New Caledonia generally exhibited smaller body measurements than those originally described from Australian waters; the deirids and eggs are described for the first time. Maricostula Bruce & Cannon, 1989 is considered a junior synonym of Hysterothylacium, to which three species are transferred as H. cenaticum (Bruce & Cannon, 1989) n. comb., H. makairi (Bruce & Cannon, 1989) n. comb. and H. tetrapteri (Bruce & Cannon, 1989) n. comb.     

The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.     

Gilquiniid cestodes (Trypanorhyncha) from elasmobranch fishes off New Caledonia with descriptions of two new genera and a new species

Cestodes were collected from deep-sea sharks caught off New Caledonia, South Pacific. Vittirhynchus squali n. g., n. sp. (Trypanorhyncha: Gilquiniidae) is described from the spiral valve of Squalus melanurus Fourmanoir & Rivaton. The new genus possesses four bothria and a typical heteroacanthous metabasal armature but has a file of three macrohooks forming a short chainette on the internal surface of the basal armature. Sagittirhynchus aculeatus n. g., n. sp., from the spiral valve of Centrophorus sp. (undescribed), also has four bothria and a typical heteroacanthous armature but lacks a distinctive basal swelling and has the final hooks of each principal row prominently enlarged. Gilquinia minor n. sp., from the spiral valve of Centrophorus sp. (undescribed), is distinguished by the presence of only five hooks per principal row compared with eight in congeners. Gilquinia sp. is reported from Squalus melanurus. G. robertsoni Beveridge, 1990 is reported from S. megalops (Macleay).