Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.     

A modified walking rhythm employed during righting behavior in the cockroachGromphadorhina portentosa

Summary Electromyograms were recorded from leg muscles of the cockroachGromphadorhina during walking and righting under free-ranging and tethered conditions. Two muscles which are essentially synergistic during walking become antagonistic during righting (Fig. 3, 4). This explains in part the difference in the direction of the leg stroke in the two behaviors (Fig. 2). Other properties of the muscle activity are very similar during the two rhythms: the same motoneurons appear to be active (Fig. 5, 6); cycle frequencies are the same; the burst length of one motoneuron studied varies with burst frequency in a generally similar manner in both behaviors (Fig. 7); inter-leg coordination is the same (Fig. 9); and transganglionic coupling characteristic of walking can occur while a leg on one side is engaged in walking, and its contralateral homologue is engaged in righting (Fig. 10). Although other properties of the leg rhythms are different in walking and righting, these differences appear to result from dissimilarities in sensory feedback. It is concluded that although the two leg rhythms are superficially quite different, the underlying central neuronal rhythms are very similar, and possibly result from activity in the same central oscillatory cell or circuit.We thank Carol Smith for technical assistance. This work was supported by NIH grant #NS09083-05. Computation was done at the New York State Veterinary College Computer Facility which is supported by NIH grant RR 326.     

A chimaeric hygromycin resistance gene as a selectable marker in plant cells

Summary We show here that plant cells are sensitive to the antibiotic hygromycin-B⁴. We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.     

Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.     

Organismal differences in post-translational modifications in histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.     

A review of the effectiveness of vaccine potency control testing

The use of potency control testing is a valuable tool for testing the actual relative strength of manufactured assembly lots of vaccine. Biological-based manufacturing methods are inherently variable and potency testing is a tool to ensure lot-to-lot consistency of commercial vaccines. A strong historical link to clinical efficacy has been established where correlation to efficacy and adequate test validation have been achieved. The link to immunogenicity and efficacy has traditionally been strongest with attenuated vaccines and toxoids. Control potency test failure does predict that a serial or batch of vaccine would most likely provide insufficient immunogenicity in typical field applications. Because of the complexity of pathogenic processes and associated immune responses, potency tests may not always directly predict the effectiveness of a vaccine. Thus, vaccines that pass control potency testing may not always provide adequate efficacy. This is particularly true of adjuvanted, inactivated vaccines. In the development of vaccine formulations and control tests for vaccines, the nature of the desired protective immune responses to the targeted pathogen (when known) should be considered. These considerations could provide better alternatives in the assays chosen as correlates of immunity and may more accurately predict efficacy and assure batch-to-batch consistency. Also, the effects of the dose and duration of antigen exposure as well as the nature of antigen presentation and generation of extrinsic cytokines could be characterised and correlated to vaccine potency as additional indicators of vaccine efficacy.     

Description and characterization of a chamber for viewing and quantifying cancer cell chemotaxis

Direct observations of cancer cell invasion underscore the importance of chemotaxis in invasion and metastasis. Yet, there is to date, no established method for real-time imaging of cancer chemotaxis towards factors clinically correlated with metastasis. A chamber has been designed and tested, called the Soon chamber, which allows the direct observation and quantification of cancer cell chemotaxis. The premise for the design of the Soon chamber is the incorporation of a dam, which creates a steep gradient while retaining stability associated with a pressure-driven system. The design is based on the characteristics of cancer cell motility such as relatively low speeds, and slower motility responses to stimuli compared to classical amoeboid cells like neutrophils and Dictyostelium. We tested MTLn3 breast carcinoma cells in the Soon chamber in the presence of an EGF gradient, obtaining hour-long time-lapses of chemotaxis. MTLn3 cells migrated further, more linearly, and at greater speeds within an EGF gradient compared to buffer controls. Computation of the degree of orientation towards the EGF/buffer source showed that MTLn3 cells were significantly more directional toward the EGF gradient compared to buffer controls. Analysis of the time-lapse data obtained during chemotaxis demonstrated that two populations of cancer cells were present. One population exhibited oscillations in directionality occurring at average intervals of 12 min while the second population exhibited sustained high levels of directionality toward the source of EGF. This result suggests that polarized cancer cells can avoid the need for oscillatory path corrections during chemotaxis.     

Effect of chlorsulfuron on ethylene evolution from sunflower seedlings

The effect of the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy - 6 - methyl -1, 3,5 - triazin - 2 - yl)aminocarbonyl]benzenesulfonamide) on ethylene production in light-grown sunflower (Helianthus annuus L.) seedlings was examined. Application of chlorsulfuron to the apex stimulated ethylene production in all tissues examined: cotyledons, hypocotyls, and roots. The greatest stimulation occurred in the upper portion of the hypocotyl adjacent to, and including, the cotyledonary node. Ethylene evolution from hypocotyls excised from treated seedlings was stimulated over control levels 1 day after herbicide application and reached a maximum (approx. 75 x control or 17 nl/g f wt/h) 2 to 3 days after treatment. Labeling and inhibitor studies indicated that the ethylene produced was derived primarily from methionine. Chlorsulfuron treatment stimulated the rate of accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as well as the ability of the tissue to convert exogenous ACC to ethylene. Chlorsulfuron had little effect on ethylene production when administered to the hypocotylsin vitro. Removal of the cotyledons from treated seedlings reduced the rate of ethylene evolution from the hypocotyls. These results suggest that stimulation of ethylene production in sunflower hypocotyls by chlorsulfuron is not a wound response but rather is dependent on factors derived from the cotyledons.