Towards synthesis of a minimal cell

Construction of a chemical system capable of replication and evolution, fed only by small molecule nutrients, is now conceivable. This could be achieved by stepwise integration of decades of work on the reconstitution of DNA, RNA and protein syntheses from pure components. Such a minimal cell project would initially define the components sufficient for each subsystem, allow detailed kinetic analyses and lead to improved in vitro methods for synthesis of biopolymers, therapeutics and biosensors. Completion would yield a functionally and structurally understood self-replicating biosystem. Safety concerns for synthetic life will be alleviated by extreme dependence on elaborate laboratory reagents and conditions for viability. Our proposed minimal genome is 113 kbp long and contains 151 genes. We detail building blocks already in place and major hurdles to overcome for completion

Simultaneous Observations of Comet C/2002 T7 (LINEAR) with the Berkeley-Illinois-Maryland Association and Owens Valley Radio Observatory Interferometers: HCN and CH_3OH

We present observations of HCN J = 1-0 and CH_3OH J(K_a, K_c) = 3(1, 3)-4(0, 4) A+ emission from comet C/2002 T7 (LINEAR) obtained simultaneously with the Owens Valley Radio Observatory (OVRO) and Berkeley-Illinois-Maryland Association (BIMA) millimeter interferometers. We combined the data from both arrays to increase the (u, v) sampling and signal to noise of the detected line emission. We also report the detection of CH_3OH J(K_a, K_c) = 8(0, 8)-7(1, 7) A^+ with OVRO data alone. Using a molecular excitation code that includes the effects of collisions with water and electrons, as well as pumping by the Solar infrared photons (for HCN alone), we find a production rate of HCN of 2.9 × 10^(26) s^(–1) and for CH_3OH of 2.2 × 10^(27) s^(–1). Compared to the adopted water production rate of 3 × 10^(29) s^(–1), this corresponds to an HCN/H_2O ratio of 0.1% and a CH_3OH/H_2O ratio of 0.7%. We critically assess the uncertainty of these values due to the noise (~10%), the uncertainties in the adopted comet model (~50%), and the uncertainties in the adopted collisional excitation rates (up to a factor of 2). Pumping by Solar infrared photons is found to be a minor effect for HCN, because our 15" synthesized beam is dominated by the region in the coma where collisions dominate. Since the uncertainties in the derived production rates are at least as large as one-third of the differences found between comets, we conclude that reliable collision rates and an accurate comet model are essential. Because the collisionally dominated region critically depends on the water production rate, using the same approximate method for different comets may introduce biases in the derived production rates. Multiline observations that directly constrain the molecular excitation provide much more reliable production rates

Planar Airy beam light-sheet for two-photon microscopy

We demonstrate the first planar Airy light-sheet microscope. Fluorescence light-sheet microscopy has become the method of choice to study large biological samples with cellular or sub-cellular resolution. The propagation-invariant Airy beam enables a ten-fold increase in field-of-view with single-photon excitation; however, the characteristic asymmetry of the light-sheet limits its potential for multi-photon excitation. Here we show how a planar light-sheet can be formed from the curved propagation-invariant Airy beam. The resulting symmetric light sheet excites two-photon fluorescence uniformly across an extended field-of-view without the need for deconvolution. We demonstrate the method for rapid two-photon imaging of large volumes of neuronal tissue.Comment: 7 pages, 4 figure

Low modularity of aminoacyl-tRNA substrates in polymerization by the ribosome

Aminoacyl-transfer RNAs contain four standardized units: amino acids, an invariant 3′-terminal CCA, trinucleotide anticodons and tRNA bodies. The degree of interchangeability of the three variable modules is poorly understood, despite its role in evolution and the engineering of translation to incorporate unnatural amino acids. Here, a purified translation system is used to investigate effects of various module swaps on the efficiency of multiple ribosomal incorporations of unnatural aminoacyl-tRNA substrates per peptide product. The yields of products containing three to five adjacent l-amino acids with unnatural side chains are low and cannot be improved by optimization or explained simply by any single factor tested. Though combinations of modules that allow quantitative single unnatural incorporations are found readily, finding combinations that enable efficient synthesis of products containing multiple unnatural amino acids is challenging. This implies that assaying multiple, as opposed to single, incorporations per product is a more stringent assay of substrate activity. The unpredictability of most results illustrates the multifactorial nature of substrate recognition and the value of synthetic biology for testing our understanding of translation. Data indicate that the degree of interchangeability of the modules of aminoacyl-tRNAs is low

A Bima Array Survey of Molecules in Comets Linear (C/2002 T7) and Neat (C/2001 Q4)

We present an interferometric search for large molecules, including methanol, methyl cyanide, ethyl cyanide, ethanol, and methyl formate in comets LINEAR (C/2002 T7) and NEAT (C/2001 Q4) with the Berkeley-Illinois-Maryland Association (BIMA) array. In addition, we also searched for transitions of the simpler molecules CS, SiO, HNC, HN13C and 13CO . We detected transitions of methanol and CS around Comet LINEAR and one transition of methanol around Comet NEAT within a synthesized beam of ~20''. We calculated the total column density and production rate of each molecular species using the variable temperature and outflow velocity (VTOV) model described by Friedel et al.(2005).Considering the molecular production rate ratios with respect to water, Comet T7 LINEAR is more similar to Comet Hale-Bopp while Comet Q4 NEAT is more similar to Comet Hyakutake. It is unclear, however, due to such a small sample size, whether there is a clear distinction between a Hale-Bopp and Hyakutake class of comet or whether comets have a continuous range of molecular production rate ratios.Comment: Accepted for Publication in the Astrophysical Journa

Molecular characterisation and genetic mapping of candidate genes for qualitative disease resistance in perennial ryegrass (Lolium perenne L.)

<p>Abstract</p> <p>Background</p> <p>Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site – leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database.</p> <p>Results</p> <p>Amplification of R gene sequences was performed using template EST data and information from orthologous candidate using a degenerate consensus PCR approach. A total of 102 unique partial R genes were cloned, sequenced and functionally annotated. Analysis of motif structure and R gene phylogeny demonstrated that <it>Lolium </it>R genes cluster with putative ortholoci, and evolved from common ancestral origins. Single nucleotide polymorphisms (SNPs) predicted through resequencing of amplicons from the parental genotypes of a genetic mapping family were validated, and 26 distinct R gene loci were assigned to multiple genetic maps. Clusters of largely non-related NBS-LRR genes were located at multiple distinct genomic locations and were commonly found in close proximity to previously mapped defence response (DR) genes. A comparative genomics analysis revealed the co-location of several candidate R genes with disease resistance quantitative trait loci (QTLs).</p> <p>Conclusion</p> <p>This study is the most comprehensive analysis to date of qualitative disease resistance candidate genes in perennial ryegrass. SNPs identified within candidate genes provide a valuable resource for mapping in various ryegrass pair cross-derived populations and further germplasm analysis using association genetics. In parallel with the use of specific pathogen virulence races, such resources provide the means to identify gene-for-gene mechanisms for multiple host pathogen-interactions and ultimately to obtain durable field-based resistance.</p

Water maser variability over 20 years in a large sample of star-forming regions: the complete database

Context. Water vapor emission at 22 GHz from masers associated with star-forming regions is highly variable. Aims. We present a database of up to 20 years of monitoring of a sample of 43 masers within star-forming regions. The sample covers a large range of luminosities of the associated IRAS source and is representative of the entire population of H2O masers of this type. The database forms a good starting point for any further study of H2O maser variability. Methods. The observations were obtained with the Medicina 32-m radiotelescope, at a rate of 4-5 observations per year. Results. To provide a database that can be easily accessed through the web, we give for each source: plots of the calibrated spectra, the velocity-time-flux density plot, the light curve of the integrated flux, the lower and upper envelopes of the maser emission, the mean spectrum, and the rate of the maser occurrence as a function of velocity. Figures for just one source are given in the text for representative purposes. Figures for all the sources are given in electronic form in the on-line appendix. A discussion of the main properties of the H2O variability in our sample will be presented in a forthcoming paper.Comment: 11 pages, 9 figures, to be published in Astronomy and Astrophysics; all plots in appendix (not included) can be downloaded from http://www.arcetri.astro.it/~starform/water_maser_v2.html or http://www.ira.inaf.it/papers/masers/water_maser_v2.htm

The Distribution, Excitation and Formation of Cometary Molecules: Methanol, Methyl Cyanide and Ethylene Glycol

We present an interferometric and single dish study of small organic species toward Comets C/1995 O1 (Hale-Bopp) and C/2002 T7 (LINEAR) using the BIMA interferometer at 3 mm and the ARO 12m telescope at 2 mm. For Comet Hale-Bopp, both the single-dish and interferometer observations of CH3OH indicate an excitation temperature of 105+/-5 K and an average production rate ratio Q(CH3OH)/Q(H2O)~1.3% at ~1 AU. Additionally, the aperture synthesis observations of CH3OH suggest a distribution well described by a spherical outflow and no evidence of significant extended emission. Single-dish observations of CH3CN in Comet Hale-Bopp indicate an excitation temperature of 200+/-10 K and a production rate ratio of Q(CH3CN)/Q(H2O)~0.017% at ~1 AU. The non-detection of a previously claimed transition of cometary (CH2OH)2 toward Comet Hale-Bopp with the 12m telescope indicates a compact distribution of emission, D<9'' (<8500 km). For the single-dish observations of Comet T7 LINEAR, we find an excitation temperature of CH3OH of 35+/-5 K and a CH3OH production rate ratio of Q(CH3OH)/Q(H2O)~1.5% at ~0.3 AU. Our data support current chemical models that CH3OH, CH3CN and (CH2OH)2 are parent nuclear species distributed into the coma via direct sublimation off cometary ices from the nucleus with no evidence of significant production in the outer coma.Comment: accepted for publication in Ap

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.

BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro

BIMA Array Detections of HCN in Comets LINEAR (C/2002 T7) and NEAT (C/2001 Q4)

We present interferometric detections of HCN in comets LINEAR (C/2002 T7) and NEAT (C/2001 Q4) with the Berkeley-Illinois-Maryland Association (BIMA) Array. With a 25.4"x20.3" synthesized beam around Comet LINEAR and using a variable temperature and outflow velocity (VTOV) model, we found an HCN column density of N_T=6.4+-2.1x10^12 cm^-2, and a production rate of Q(HCN)=6.5+-2.2x10^26 s^-1, giving a production rate ratio of HCN relative to H_2O of ~3.3+-1.1x10^-3 and relative to CN of ~4.6+-1.5. With a 21.3"x17.5" synthesized beam around Comet NEAT and using a VTOV model, we found an HCN column density of N_T=8.5+-4.5x10^11 cm^-2, and a production rate of Q(HCN)=8.9+-4.7x10^25 s^-1, giving a production rate ratio of HCN relative to H_2O of ~7.4+-3.9x10^-4 and relative to CN of ~0.3+-0.2. For both comets, the production rates relative to H_2O are similar to those found in previous comet observations. For Comet LINEAR the production rate relative to CN is consistent with HCN being the primary parent species of CN, while for Comet NEAT it is too low for this to be the case.Comment: 20 pages, 2 figures, to be published in the Astrophysical Journa