Temperature-Sensitive, Assembly-Defective Flagella Mutants of CHLAMYDOMONAS REINHARDTII

We describe an efficient selection procedure for the isolation of mutants of Chlamydomonas reinhardtii with temperature sensitive flagella defects, with final yields of up to 11% of the population being mutant. Several mutants, all showing an inability to maintain flagellar integrity at the restrictive temperature, are described. We have examined flagellar stability and reassembly at various temperatures in the mutants. Mapping data are provided for these, as well as for some previously described mutants.     

A putative ABC-transport operon of Mycobacterium smegmatis

We have recently described the mpr gene of Mycobacterium smegmatis whose product confers resistance to mycobacteriophages L5 and D29 when overproduced (Barsom and Hatfull (1996) Mol. Microbiol. 21, 159–170). We have determined the nt sequence of approximately 3.5 kb immediately adjacent to mpr which appears to encode components of an ATP-binding cassette (ABC) transport system. Four closely-spaced open reading frames (ORF) were identified although two of these may cooperate to produce an integral membrane component of the transport system via a programmed translational frameshift. Another putative protein is also predicted to be an integral membrane protein, while the third is an ABC-transporter protein. We propose that these three putative proteins form a mycobacterial membrane-bound complex involved in protein-dependent transport. This is the first ABC-transport system to be described in mycobacteria.     

SDF-1 Chemokine Signalling Modulates the Apoptotic Responses to Iron Deprivation of Clathrin-Depleted DT40 Cells

We have previously deleted both endogenous copies of the clathrin heavy-chain gene in the chicken pre B-cell-line DT40 and replaced them with clathrin under the control of a tetracycline-regulatable promoter (Tet-Off). The originally derived cell-line DKO-S underwent apoptosis when clathrin expression was repressed. We have also described a cell-line DKO-R derived from DKO-S cells that was less sensitive to clathrin-depletion. Here we show that the restriction of transferrin uptake, resulting in iron deprivation, is responsible for the lethal consequence of clathrin-depletion. We further show that the DKO-R cells have up-regulated an anti-apoptotic survival pathway based on the chemokine SDF-1 and its receptor CXCR4. Our work clarifies several puzzling features of clathrin-depleted DT40 cells and reveals an example of how SDF-1/CXCR4 signalling can abrogate pro-apoptotic pathways and increase cell survival. We propose that the phenomenon described here has implications for the therapeutic approach to a variety of cancers.     

Primary Neuropathic Amyloidosis in Three Brothers

Neuropathic primary amyloidosis has not been previously described in an English family. We present the clinical and pathological findings in three brothers with this condition and suggest that they have suffered from that form of the disease described originally by Andrade.     

Measurement of arachidonic acid in the plasma by gas-liquid chromatography-flame ionization using dihomo-gamma-linolenic acid as an internal standard.

A gas-liquid chromatography-flame ionization method is described for measuring arachidonic acid in plasma using dihomo-gamma-linolenic acid as an internal standard. We found this technique to -e reproducible, and quicker and superior to previously described techniques because of the similar physico-chemical properties of the unsaturated fatty acid internal standard and arachidonic acid. In addition, we observed that the use of the saturated fatty acid, n-tricosanoic acid, was unsatisfactory as an internal standard because of its poor extractability from plasma as compared to arachidonic acid.     

pRRophetic: An R Package for Prediction of Clinical Chemotherapeutic Response from Tumor Gene Expression Levels

We recently described a methodology that reliably predicted chemotherapeutic response in multiple independent clinical trials. The method worked by building statistical models from gene expression and drug sensitivity data in a very large panel of cancer cell lines, then applying these models to gene expression data from primary tumor biopsies. Here, to facilitate the development and adoption of this methodology we have created an R package called pRRophetic. This also extends the previously described pipeline, allowing prediction of clinical drug response for many cancer drugs in a user-friendly R environment. We have developed several other important use cases; as an example, we have shown that prediction of bortezomib sensitivity in multiple myeloma may be improved by training models on a large set of neoplastic hematological cell lines. We have also shown that the package facilitates model development and prediction using several different classes of data.     

Statistical inference for familial disease clusters

In many epidemiologic studies, the first indication of an environmental or genetic contribution to the disease is the way in which the diseased cases cluster within the same family units. The concept of clustering is contrasted with incidence. We assume that all individuals are exchangeable except for their disease status. This assumption is used to provide an exact test of the initial hypothesis of no familial link with the disease, conditional on the number of diseased cases and the distribution of the sizes of the various family units. New parametric generalizations of binomial sampling models are described to provide measures of the effect size of the disease clustering. We consider models and an example that takes covariates into account. Ascertainment bias is described and the appropriate sampling distribution is demonstrated. Four numerical examples with real data illustrate these methods.     

Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker

Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.     

Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids.     

A new kind of prion: a modified protein necessary for its own modification

We recently described an infectious protein (prion) unrelated to amyloid formation, that is an enzyme whose precursor can only be activated by the active form of the enzyme. All previously described infectious proteins are self-propagating amyloid forms of chromosomally encoded proteins. The infectious enzyme, vacuolar protease B (PrB), can activate its own precursor in an indefinitely self-propagating process. Transfer from cell to cell of cytoplasm containing active protease B transmits this non-chromosomal gene. The importance of this system is that many protein-modifying enzymes may act on themselves, and if conditions are right, may become prions as well.     

Incorporation of carbon from decomposing litter of two pioneer plant species into microbial communities of the detritusphere

For several Staphylococci, such as Staphylococcus aureus, Staphylococcus saprophyticus, and Staphylococcus epidermidis, invasion of eukaryotic cells has been described and this mechanism has been considered an important part of the infection process. The fibrinogen-binding protein (Fbl) of Staphylococcus lugdunensis, a homolog of the clumping factor A of S. aureus, has been described as fibrinogen-binding adhesin and might promote invasion of cells. We therefore characterized several clinical strains of S. lugdunensis in terms of whole cell fibrinogen and fibronectin binding and correlated these results with the invasion of epithelial and endothelial cells by S. lugdunensis. We described for the first time invasion of cells by S. lugdunensis. As invasion of cells by S. lugdunensis was only partly inhibited by cytochalasin D in contrast to a complete inhibition of invasion of cells by S. aureus, further invasion mechanisms are likely to be present in S. lugdunensis. In addition, the Fbl of S. lugdunensis is not involved in the invasion of cells as ruled out by an isogenic fbl mutant.     

Where to sit in a waiting room: density, age and gender effects on proxemic choices

We studied proxemic behaviour in an hospital waiting room by recording who chose to stand and who chose to sit and which chair, row and position. We distinguished sex and two categories of age, mature and old. We found that fenales sit together more easily than males and more frequently chose to sit close, especially mature ones. Males tend to keep more distances from neighbours by standing up. In general, proxemic behaviour in a waiting room seems to be an indicator confirming the sexual startegies in partner choice described in the evolutionary psychology paradigm.     

Ligand trapping in epithelial layers and cell cultures

We analyze a stochastic model that describes receptor-mediated ligand trapping in epithelial layers and cell culture assays. In both cases, the problem is reduced to diffusion of a Brownian particle between the partially absorbing and reflective surfaces. We derive an analytical expression for the spatial distribution of the trapping points and identify the domains of applicability of the two limiting regimes. We conclude that a thin layer approximation is applicable for ligand trapping in epithelial layers while a typical cell culture experiment is appropriately described within an infinite layer approximation.     

Landscape‐scale determinants of non‐native fish communities

Aim Predicting and preventing invasions depends on knowledge of the factors that make ecosystems susceptible to invasion. Current studies generally rely on non‐native species richness (NNSR) as the sole measure of ecosystem invasibility; however, species identity is a critical consideration, given that different ecosystems may have environmental characteristics suitable to different species. Our aim was to examine whether non‐native freshwater fish community composition was related to ecosystem characteristics at the landscape scale. Location United States. Methods We described spatial patterns in non‐native freshwater fish communities among watersheds in the Mid‐Atlantic region of the United States based on records of establishment in the U.S. Geological Survey’s Nonindigenous Aquatic Species Database. We described general relationships between non‐native species and ecosystem characteristics using canonical correspondence analysis. We clustered watersheds by non‐native fish community and described differences among clusters using indicator species analysis. We then assessed whether non‐native communities could be predicted from ecosystem characteristics using random forest analysis and predicted non‐native communities for uninvaded watersheds. We estimated which ecosystem characteristics were most important for predicting non‐native communities using conditional inference trees. Results We identified four non‐native fish communities, each with distinct indicator species. Non‐native communities were predicted based on ecosystem characteristics with an accuracy of 80.6%, with temperature as the most important variable. Relatively uninvaded watersheds were predicted to be invasible by the most diverse non‐native community. Main conclusions Non‐native species identity is an important consideration when assessing ecosystem invasibility. NNSR alone is an insufficient measure of invasibility because ecosystems with equal NNSR may not be equally invasible by the same species. Our findings can help improve predictions of future invasions and focus management and policy decisions on particular species in highly invasible ecosystems.     

Nucleotide excision repair in chromatin: damage removal at the drop of a HAT

In an earlier review of our understanding of the mechanism of nucleotide excision repair (NER) we examined the process with respect to how it occurs in chromatin [1]. We described how much of our mechanistic understanding of NER was derived from biochemical studies that analysed the repair reaction in DNA substrates not representative of that which exists in the living cell. We pointed out that our efforts to understand how NER operates in chromatin had been hampered in part because of the well-known inhibition of NER that occurs when DNA is assembled into nucleosomes and used as the substrate to examine the repair reaction in vitro. Despite this technical bottleneck, we summarized the biochemical, genetic and cell-based studies which have provided insights into the molecular mechanism of NER in the cellular context. More recently, we revisited the topic of how UV induced DNA damage is repaired in chromatin. In this review we examined the commonly held view that depicts a struggle in which the DNA repair machinery battles to overcome the inhibitory effect of chromatin during the repair process. We suggested that in this interpretation of events, the DNA repair mechanisms might be described as 'tilting at windmills': fighting an imaginary foe [2]. We surmised that this scenario was overly simplistic, and we described an emerging picture in which the DNA repair process and chromatin remodeling were mechanistically linked and were in fact functioning cooperatively to organize the efficient removal of DNA damage from the genome. Here we discuss the latest findings, which contribute to the idea that DNA damage induced changes to chromatin represent an important way in which the DNA repair process is initiated and organized throughout the genome to promote the efficient removal of damage in response to UV radiation.     

A modified scoring system to describe gross pathology in the rabbit model of tuberculosis

Background  

The rabbit model is an ideal means to study the pathogenesis of tuberculosis due to its semblance to the disease in humans. We have previously described the results using a bronchoscopic route of infection with live bacilli as a reliable means of generating lung cavities in sensitized rabbits. The role of sensitization in the development of disease outcomes has been well established in several animal models. We have described here the varying gross pathology that result from lack of sensitization with heat-killed M. bovis prior to high-dose bronchoscopic infection with live bacilli.     

The temporal island scalp flap for management of facial burn scars

Facial burn scars are difficult to conceal and often preclude an aesthetic rehabilitation of the patient. Multistaged scalp and neck flaps have been described to provide hair-bearing skin to resurface burn scars in men. We have been resurfacing the upper lip and cheek in a one-stage procedure using a temporal artery island scalp flap. The temporoparietal fascia has been well described in recent years, and the understanding of this anatomy has facilitated the use of the island scalp flap for more distal transfers.     

Aproximating static list schedules in dynamic multithreaded applications

List scheduling algorithms are known to be efficient when the application to be executed can be described statically as a Directed Acyclic Graph (DAG) of tasks. Regardless of knowing the entire DAG beforehand, obtaining an optimal schedule in a parallel machine is a NP-hard problem. Moreover, many programming tools propose the use of scheduling techniques based on list strategies. This paper presents an analysis of scheduling algorithms for multithread programs in a dynamic scenario where threads are created and destroyed during execution. We introduce an algorithm to convert DAGs, describing applications as tasks, into Directed Cyclic Graphs (DCGs) describing the same application designed in a multithread programming interface. Our algorithm covers case studies described in previous works, successfully mapping from the abstract level of graphs to the application environment. These mappings preserve the guarantees offered by the abstract model, providing efficient scheduling of dynamic programs that follow the intended multithread model. We conclude the paper presenting some performance results we obtained by list schedulers in dynamic multithreaded environments. We also compare these results with the best scheduling we could obtain with similar static task schedulers.