The genetic basis of alcoholism: multiple phenotypes,many genes,complex networks

Alcoholism is a significant public health problem. A picture of the genetic architecture underlying alcohol-related phenotypes is emerging from genome-wide association studies and work on genetically tractable model organisms.     

Competition in vitro among fungi acquired from the exoskeleton of the giant Madagascar hissing-cockroach,Gromphadorhina portentosa,and its relevance to human health

Few insects have received as much public attention as the Madagascar hissing-cockroach, Gromphadorhina portentosa, coveted by children as a household pet. Recently, this insect has been linked to allergies and asthma triggered by an antagonistic external mycoflora, and some of these fungi are known to cause serious illnesses, namely Aspergillus spp., Mucor spp. and Rhizopus spp. Other cockroach species serve as carriers of medically important fungi, but G. portentosa is of special concern because it is deliberately reared in captivity. The aim of this study was to ascertain why certain types of fungi dominate the exoskeleton’s mycoflora whereas others do not. Six ascomycetes (Alternaria alternata, Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides, Penicillium glabrum, Trichoderma viride), and two zygomycetes (Mucor racemosus, Rhizopus stolonifer) initially acquired from the exoskeleton of G. portentosa, were tested for their competitive ability to inhibit one another in vitro, using the cockroach as the primary substratum. R. stolonifer, M. racemosus, and mycoparasitic T. viride grew at a significantly faster rate compared to the other taxa, and growth rates correlated positively with conidia output. Slower colony growth rates were noted prior to physical contact, suggesting that the fungi engaged in chemical antagonism. Three genera emerged that best fit the profile of exploitative competitors – Rhizopus, Mucor, Trichoderma. The latter two genera completely inhibited the growth of all fungal taxa tested and also had some capacity to grow over the mycelium of another fungus. We conclude that M. racemosus and T. viride in particular, are successful agents of G. portentosa’s mycoflora because they utilize both exploitation and interference competition to their advantage. Eliminating or reducing the simple sugar load entering the cockroach colony may be one way to tilt the composition of the mycoflora to favour strains that are less medically significant. Thus, we emphasize reduction of the antagonistic fungi at the colony (food entry) level and less so on the exoskeleton level.     

Membrane Damage and Microbial Inactivation by Chlorine in the Absence and Presence of a Chlorine-Demanding Substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.     

RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

Background  

The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures.     

Novel and efficient RNA secondary structure prediction using hierarchical folding.

Algorithms for prediction of RNA secondary structure-the set of base pairs that form when an RNA molecule folds-are valuable to biologists who aim to understand RNA structure and function. Improving the accuracy and efficiency of prediction methods is an ongoing challenge, particularly for pseudoknotted secondary structures, in which base pairs overlap. This challenge is biologically important, since pseudoknotted structures play essential roles in functions of many RNA molecules, such as splicing and ribosomal frameshifting. State-of-the-art methods, which are based on free energy minimization, have high run-time complexity (typically Theta(n(5)) or worse), and can handle (minimize over) only limited types of pseudoknotted structures. We propose a new approach for prediction of pseudoknotted structures, motivated by the hypothesis that RNA structures fold hierarchically, with pseudoknot-free (non-overlapping) base pairs forming first, and pseudoknots forming later so as to minimize energy relative to the folded pseudoknot-free structure. Our HFold algorithm uses two-phase energy minimization to predict hierarchically formed secondary structures in O(n(3)) time, matching the complexity of the best algorithms for pseudoknot-free secondary structure prediction via energy minimization. Our algorithm can handle a wide range of biological structures, including kissing hairpins and nested kissing hairpins, which have previously required Theta(n(6)) time.     

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of low dissolved oxygen on survival and asexual reproduction of scyphozoan polyps (Chrysaora quinquecirrha)

Hypoxic conditions are common in many coastal environments such as Chesapeake Bay. While medusae appear to be quite tolerant of low dissolved oxygen (DO) concentrations, the effects of hypoxia on the benthic polyp stages are unknown. Chrysaora quinquecirrha (DeSor) polyps, and were subjected to 5 DO treatments (air-saturated [control], 3.5, 2.5, 1.5 and 0.5 mg l^–1) in the laboratory. Polyp survival and development were documented over 24 d. Virtually no mortality occurred in any treatment during the first 5 d. Total polyp mortality after 24 d was 59.3% at the lowest DO concentration, whereas <3% mortality was observed in the air-saturated treatment. Formation of stolons and strobilae occurred in all treatments, however, the proportions of polyps undergoing stolonation and strobilation were significantly greater in all DO concentrations above 0.5 mg l^–1. Polyp encystment was not observed in any treatment over the course of the 24 d experiment. These results indicate that polyps can survive and asexually propagate even during prolonged exposure to hypoxic conditions.