A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain

Using RNAi screening, proteomics, cell biological and mouse genetics approaches, we have identified a complex of nine proteins, seven of which are disrupted in human ciliopathies. A transmembrane component, TMEM231, localizes to the basal body before and independently of intraflagellar transport in a Septin 2 (Sept2)-regulated fashion. The localizations of TMEM231, B9D1 (B9 domain-containing protein 1) and CC2D2A (coiled-coil and C2 domain-containing protein 2A) at the transition zone are dependent on one another and on Sept2. Disruption of the complex in vitro causes a reduction in cilia formation and a loss of signalling receptors from the remaining cilia. Mouse knockouts of B9D1 and TMEM231 have identical defects in Sonic hedgehog (Shh) signalling and ciliogenesis. Strikingly, disruption of the complex increases the rate of diffusion into the ciliary membrane and the amount of plasma-membrane protein in the cilia.?The complex that we have described is essential for normal cilia function and acts as a diffusion barrier to maintain the cilia membrane as a compartmentalized signalling organelle.     

A Detailed, Hierarchical Study of Giardia lamblia's Ventral Disc Reveals Novel Microtubule-Associated Protein Complexes

Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia''s two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.     

Parents' perceptions of food intolerance in primary school children.

In a study of about 7000 children, parents'' perceptions were used to examine the prevalence of food intolerance, the types of food implicated, the association of intolerance with diseases, and the social background of those identified as being food intolerant. One hundred and ninety two children (3%) were perceived as being food intolerant, with a further 105 (2%) being classed as intolerant under a less stringent definition of intolerance. For 128 (67%) of these children a doctor was consulted. The pattern of food avoided was very similar in children for whom the decision to exclude certain foods was made by health staff and in those for whom the parents themselves made decisions about their child''s diet. A strong association was seen between the mother''s level of education and the child being perceived as being food intolerant. Between 20% and 30% of children with a disease associated with food intolerance--for example, eczema--had currently or previously avoided some types of food. The results of this study emphasise the need to develop criteria to tackle the growing demand for National Health Service treatment by parents who believe their child to be food intolerant.     

Microbial pretreatment of cotton stalks by submerged cultivation of Phanerochaete chrysosporium

This study used the fungus, Phanerochaete chrysosporium, to pretreat cotton stalks with two methods, shallow stationary and agitated cultivation, at three supplemental salt concentrations. Pretreatment efficiencies were compared by evaluating lignin degradation, solid recovery and carbohydrate availability over a 14-day period. Shallow stationary cultivation with no salts gave 20.7% lignin degradation along with 76.3% solid recovery and 29.0% carbohydrate availability. The highest lignin degradation of 33.9% at a corresponding solid recovery and carbohydrate availability of 67.8% and 18.4%, respectively, was obtained through agitated cultivation with Modified NREL salts. Cultivation beyond 10 days did not significantly increase lignin degradation during 14 days of pretreatment. Manganese addition during shallow stationary and agitated cultivation resulted in higher solid recoveries of over 80% but lower lignin degradation. Although agitated cultivation resulted in better delignification, results indicate that pretreatment under submerged shallow stationary conditions provides a better balance between lignin degradation and carbohydrate availability.     

Characterization of Clostridium ljungdahlii OTA1: a non-autotrophic hyper ethanol-producing strain

Applied Microbiology and Biotechnology - A Clostridium ljungdahlii lab-isolated spontaneous-mutant strain, OTA1, has been shown to produce twice as much ethanol as the C. ljungdahlii ATCC 55383...     

Microbial pretreatment of cotton stalks by solid state cultivation of Phanerochaete chrysosporium

White rot fungi degrade lignin and have biotechnological applications in conversion of lignocellulose to valuable products. Pretreatment is an important processing step to increase the accessibility of cellulosic material in plant biomass, impacting efficiency of subsequent hydrolysis and fermentation. This study investigated microbial pretreatment of cotton stalks by solid state cultivation (SSC) using Phanerochaete chrysosporium to facilitate the conversion into ethanol. The effects of substrate moisture content (M.C.; 65%, 75% and 80% wet-basis), inorganic salt concentration (no salts, modified salts without Mn(2+), modified salts with Mn(2+)) and culture time (0-14 days) on lignin degradation (LD), solids recovery (SR) and availability of carbohydrates (AOC) were examined. Moisture content significantly affected lignin degradation, with 75% and 80% M.C. degrading approximately 6% more lignin than 65% M.C. after 14 days. Within the same moisture content, treatments supplemented with salts were not statistically different than those without salts for LD and AOC. Within the 14day pretreatment, additional time resulted in greater lignin degradation, but indicated a decrease in SR and AOC. Considering cost, solid state cultivation at 75% M.C. without salts was the most preferable pretreatment resulting in 27.6% lignin degradation, 71.1% solids recovery and 41.6% availability of carbohydrates over a period of 14 days. Microbial pretreatment by solid state cultivation has the potential to be a low cost, environmentally friendly alternative to chemical approaches. Moisture relationships will be significant to the design of an effective microbial pretreatment process using SSC technology.     

Adaptive radiation within New Zealand endemic species of the cockroach genus Celatoblatta Johns (Blattidae): a response to Plio-Pleistocene mountain building and climate change

The South Island of New Zealand offers unique opportunities to study insect evolution due to long-term physical isolation, recent alpine habitats and high levels of biotic endemism. Using DNA sequence data from cytochrome oxidase subunit 1, we investigated the phylogeographical pattern among 10 endemic cockroach species within the genus Celatoblatta Johns (Blattidae). We tested the hypothesis that an ancestral cockroach species underwent rapid speciation in response to major climatic differentiation induced by mountain building. Results suggest that speciation was a twofold process, with an interspecific radiation of Pliocene/Pleistocene age followed by intraspecific diversification during the mid Pleistocene. Average genetic distance (maximum likelihood GTR + I + Gamma) was 9.17%, with a maximum of 14.5%. Data revealed eight deep well-supported branches, each with terminal clades. Six clades were differentiated according to morphological species, while the seventh was composed of three sympatric species. We consider the latter to be a phylogenetic species, possibly as a result of hybridization within a defined geographical area. This finding seriously challenges species distinctions for these three cockroach species. Correlation between genetic distances and a Climate Similarity Index (CSI) was negative, suggesting that species found in similar habitats are also genetically closely related. A Mantel test on within-clade genetic distances vs. linear geographical distance was positive, suggesting allopatric isolation for those haplotypes. We present a model of speciation for South Island Celatoblatta.     

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5(') nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations(1). This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.     

Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations

The concept of stem-cell plasticity received strong support from a recent observation that extensively passaged, clonally derived neural stem cells could contribute to hematopoiesis. We investigated whether hematopoietic potential was a consistent or unusual feature of neural stem cells, and whether it depended on the extent of in vitro passaging before transplantation. Here we transplanted over 128 x 10(6) neurosphere cells into 128 host animals; however, we never observed contribution to hematopoiesis, irrespective of the number of passages and despite the use of an assay that could detect the contribution of a single blood stem cell to hematopoietic repopulation. Although extensively cultured neurosphere cells continued to generate neural progeny, marked changes in their growth properties occurred, including changes in growth-factor dependence, cell-cycle kinetics, cell adhesion and gene expression. Our results exclude hematopoietic competence as a consistent property of intravenously infused neural stem cells. However, the consistent changes that occurred during extended passaging are compatible with genetic or epigenetic alterations and suggest that rare transformation events may account for the neural-to-blood fate switch originally reported.