Hazards in the use of Cibacron Blue F3GA in studies of proteins.

Cibacron Blue F3GA from several commercial sources is shown to be heterogeneous. This crude dye inactivates both phosphoglycerate kinase and isoleucyl-tRNA synthetase. Purification of Cibacron Blue F3GA to homogeneity results in a dramatic decrease in inactivation of these enzymes. The inactivation is shown to be due to covalent modification of phosphoglycerate kinase and probably isoleucyl-tRNA synthetase by a minor component present in crude Cibacron Blue F3GA.     

Univariate Community Assembly Analysis (UniCAA): Combining hierarchical models with null models to test the influence of spatially restricted dispersal,environmental filtering,and stochasticity on community assembly

Identifying the influence of stochastic processes and of deterministic processes, such as dispersal of individuals of different species and trait‐based environmental filtering, has long been a challenge in studies of community assembly. Here, we present the Univariate Community Assembly Analysis (UniCAA) and test its ability to address three hypotheses: species occurrences within communities are (a) limited by spatially restricted dispersal; (b) environmentally filtered; or (c) the outcome of stochasticity—so that as community size decreases—species that are common outside a local community have a disproportionately higher probability of occurrence than rare species. The comparison with a null model allows assessing if the influence of each of the three processes differs from what one would expect under a purely stochastic distribution of species. We tested the framework by simulating “empirical” metacommunities under 15 scenarios that differed with respect to the strengths of spatially restricted dispersal (restricted vs. not restricted); habitat isolation (low, intermediate, and high immigration rates); and environmental filtering (strong, intermediate, and no filtering). Through these tests, we found that UniCAA rarely produced false positives for the influence of the three processes, yielding a type‐I error rate ≤5%. The type‐II error rate, that is, production of false negatives, was also acceptable and within the typical cutoff (20%). We demonstrate that the UniCAA provides a flexible framework for retrieving the processes behind community assembly and propose avenues for future developments of the framework.     

The invertebrate fauna of High Arctic seabird nests: the microarthropod community inhabiting nests on Spitsbergen,Svalbard

The invertebrate fauna of the nests of three seabird species, black-legged kittiwakes (Rissa tridactyla), common eider (Somateria mollissima) and glaucous gull (Larus hyperboreus), were sampled in Kongsfjorden, Spitsbergen, Svalbard. The invertebrate community was species poor, consisting predominantly of the flea, Mioctenopsylla arctica arctica (Insecta: Siphonaptera), but with six species of oribatid mite (Acari: Oribatida), Diapterobates notatus, Oribatula tibialis, Ameronothrus lineatus, Hermannia reticulata, Trichoribates trimaculatus and Ceratoppia bipilis, plus an occasional mesostigmatid mite. No Collembola or ticks (Acari: Ixodidae) were observed. With the exception of M. arctica arctica, the fauna of seabird nests consisted of opportunistic microarthropod species rather than specialised nest-dwelling or bird parasitic species. Species diversity of soil oribatid mites was greater in nests of the common eider than compared to nests of the black-legged kittiwake, which may be related to the ground nesting behaviour of the common eiders. No rare or unusual microarthropod species for Svalbard were found in the seabird nests. The contentions that nests may facilitate microarthropod colonisation of High Arctic regions via bird phoresy by providing a high-quality habitat at the point of arrival, or that there might be a specialised microarthropod fauna exploiting this habitat, were not supported in this study. These are amongst the first data on the microarthropod community of seabird nests in the High Arctic.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Biodegradation of volatile organic compounds by five fungal species

Five fungal species, Cladosporium resinae (ATCC 34066), Cladosporium sphaerospermum (ATCC 200384), Exophiala lecanii-corni (CBS 102400), Mucor rouxii (ATCC 44260), and Phanerochaete chrysosporium (ATCC 24725), were tested for their ability to degrade nine compounds commonly found in industrial off-gas emissions. Fungal cultures inoculated on ceramic support media were provided with volatile organic compounds (VOCs) via the vapor phase as their sole carbon and energy sources. Compounds tested included aromatic hydrocarbons (benzene, ethylbenzene, toluene, and styrene), ketones (methyl ethyl ketone, methyl isobutyl ketone, and methyl propyl ketone), and organic acids ( n-butyl acetate, ethyl 3-ethoxypropionate). Experiments were conducted using three pH values ranging from 3.5 to 6.5. Fungal ability to degrade each VOC was determined by observing the presence or absence of visible growth on the ceramic support medium during a 30-day test period. Results indicate that E. lecanii-corni and C. sphaerospermum can readily utilize each of the nine VOCs as a sole carbon and energy source. P. chrysosporium was able to degrade all VOCs tested except for styrene under the conditions imposed. C. resinae was able to degrade both organic acids, all of the ketones, and some of the aromatic compounds (ethylbenzene and toluene); however, it was not able to grow utilizing benzene or styrene under the conditions tested. With the VOCs tested, M. rouxiiproduced visible growth only when supplied with n-butyl acetate or ethyl 3-ethoxypropionate. Maximum growth for most fungi was observed at a pH of approximately 5.0. The experimental protocol utilized in these studies is a useful tool for assessing the ability of different fungal species to degrade gas-phase VOCs under conditions expected in a biofilter application.     

Avidin is a heparin-binding protein. Affinity,specificity and structural analysis

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.     

Stem cells from the adult human brain develop into functional neurons in culture

Recent research communications indicate that the adult human brain contains undifferentiated, multipotent precursors or neural stem cells. It is not known, however, whether these cells can develop into fully functional neurons. We cultured cells from the adult human ventricular wall as neurospheres and passed them at the individual cell level to secondary neurospheres. Following dissociation and plating, the cells developed the antigen profile of the three main cell types in the brain (GFAP, astrocytes; O2, oligodendrocytes; and beta-III-tubulin/NeuN, neurons). More importantly, the cells developed the electrophysiological profiles of neurons and glia. Over a period of 3 weeks, neuron-like cells went through the same phases as neurons do during development in vivo, including up-regulation of inward Na+ -currents, drop in input resistance, shortening of the action potential, and hyperpolarization of the cell membrane. The cells developed overshooting action potentials with a mature configuration. Recordings in voltage-clamp mode displayed both the fast inactivating TTX-sensitive sodium current (INa) underlying the rising phase of the action potential and the two potassium currents terminating the action potential in mature neurons (IA and IK, sensitive to 4-AP and TEA, respectively). We have thus demonstrated that the human ventricular wall contains multipotent cells that can differentiate into functionally mature neurons.     

Functional design of bacterial mechanosensitive channels. Comparisons and contrasts illuminated by random mutagenesis

MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression of Escherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.     

Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons

The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.     

Correlating a protein structure with function of a bacterial mechanosensitive channel

MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 A. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. coli membranes and azolectin proteoliposomes. Furthermore, we show that M. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.