Site-Specific One-to-One Click Coupling of Single Proteins to Individual Carbon Nanotubes: A Single-Molecule Approach.

We report the site-specific coupling of single proteins to individual carbon nanotubes (CNTs) in solution and with single-molecule control. Using an orthogonal Click reaction, Green Fluorescent Protein (GFP) was engineered to contain a genetically encoded azide group and then bound to CNT ends in different configurations: in close proximity or at longer distances from the GFP's functional center. Atomic force microscopy and fluorescence analysis in solution and on surfaces at the single-protein level confirmed the importance of bioengineering optimal protein attachment sites to achieve direct protein-nanotube communication and bridging.

Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium.

BACKGROUND: Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. RESULTS: We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. CONCLUSIONS: Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract.

Probing the active fraction of soil microbiomes using BONCAT-FACS.

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.

Functional analysis of maize RAD51 in meiosis and double-strand break repair.

In Saccharomyces cerevisiae, Rad51p plays a central role in homologous recombination and the repair of double-strand breaks (DSBs). Double mutants of the two Zea mays L. (maize) rad51 homologs are viable and develop well under normal conditions, but are male sterile and have substantially reduced seed set. Light microscopic analyses of male meiosis in these plants reveal reduced homologous pairing, synapsis of nonhomologous chromosomes, reduced bivalents at diakinesis, numerous chromosome breaks at anaphase I, and that >33% of quartets carry cells that either lack an organized nucleolus or have two nucleoli. This indicates that RAD51 is required for efficient chromosome pairing and its absence results in nonhomologous pairing and synapsis. These phenotypes differ from those of an Arabidopsis rad51 mutant that exhibits completely disrupted chromosome pairing and synapsis during meiosis. Unexpectedly, surviving female gametes produced by maize rad51 double mutants are euploid and exhibit near-normal rates of meiotic crossovers. The finding that maize rad51 double mutant embryos are extremely susceptible to radiation-induced DSBs demonstrates a conserved role for RAD51 in the repair of mitotic DSBs in plants, vertebrates, and yeast.

Belowground Response to Drought in a Tropical Forest Soil. II. Change in Microbial Function Impacts Carbon Composition.

Climate model projections for tropical regions show clear perturbation of precipitation patterns leading to increased frequency and severity of drought in some regions. Previous work has shown declining soil moisture to be a strong driver of changes in microbial trait distribution, however, the feedback of any shift in functional potential on ecosystem properties related to carbon cycling are poorly understood. Here we show that drought-induced changes in microbial functional diversity and activity shape, and are in turn shaped by, the composition of dissolved and soil-associated carbon. We also demonstrate that a shift in microbial functional traits that favor the production of hygroscopic compounds alter the efflux of carbon dioxide following soil rewetting. Under drought the composition of the dissolved organic carbon pool changed in a manner consistent with a microbial metabolic response. We hypothesize that this microbial ecophysiological response to changing soil moisture elevates the intracellular carbon demand stimulating extracellular enzyme production, that prompts the observed decline in more complex carbon compounds (e.g., cellulose and lignin). Furthermore, a metabolic response to drought appeared to condition (biologically and physically) the soil, notably through the production of polysaccharides, particularly in experimental plots that had been pre-exposed to a short-term drought. This hysteretic response, in addition to an observed drought-related decline in phosphorus concentration, may have been responsible for a comparatively modest CO2 efflux following wet-up in drought plots relative to control plots.

Resolving brain regions using nanostructure initiator mass spectrometry imaging of phospholipids.

In a variety of neurological diseases, pathological progression is cell type and region specific. Previous reports suggest that mass spectrometry imaging has the potential to differentiate between brain regions enriched in specific cell types. Here, we utilized a matrix-free surface mass spectrometry approach, nanostructure initiator mass spectrometry (NIMS), to show that spatial distributions of multiple lipids can be used as a 'fingerprint' to discriminate between neuronal- and glial- enriched brain regions. In addition, glial cells from different brain regions can be distinguished based on unique lipid profiles. NIMS images were generated from sagittal brain sections and were matched with immunostained serial sections to define glial cell enriched areas. Tandem mass spectrometry (LC-MS/MS QTOF) on whole brain extracts was used to identify 18 phospholipids. Multivariate statistical analysis (Nonnegative Matrix Factorization) enhanced differentiation of brain regions and cell populations compared to single ion imaging methods. This analysis resolved brain regions that are difficult to distinguish using conventional stains but are known to have distinct physiological functions. This method accurately distinguished the frontal (or somatomotor) and dorsal (or retrosplenial) regions of the cortex from each other and from the pons region.

Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas).

Compared with understanding of biological shape and form, knowledge is sparse regarding what regulates growth and body size of a species. For example, the genetic and physiological causes of heterosis (hybrid vigor) have remained elusive for nearly a century. Here, we investigate gene-expression patterns underlying growth heterosis in the Pacific oyster (Crassostrea gigas) in two partially inbred (f = 0.375) and two hybrid larval populations produced by a reciprocal cross between the two inbred families. We cloned cDNA and generated 4.5 M sequence tags with massively parallel signature sequencing. The sequences contain 23,274 distinct signatures that are expressed at statistically nonzero levels and show a highly positively skewed distribution with median and modal counts of 9.25 million and 3 transcripts per million, respectively. For nearly half of these signatures, expression level depends on genotype and is predominantly nonadditive (hybrids deviate from the inbred average). Statistical contrasts suggest approximately 350 candidate genes for growth heterosis that exhibit concordant nonadditive expression in reciprocal hybrids; this represents only approximately 1.5% of the >20,000 transcripts. Patterns of gene expression, which include dominance for low expression and even underdominance of expression, are more complex than predicted from classical dominant or overdominant explanations of heterosis. Preliminary identification of ribosomal proteins among candidate genes supports the suggestion from previous studies that efficiency of protein metabolism plays a role in growth heterosis.

Genome-wide transcript analysis of maize hybrids: allelic additive gene expression and yield heterosis.

Heterosis, or hybrid vigor, has been widely exploited in plant breeding for many decades, but the molecular mechanisms underlying the phenomenon remain unknown. In this study, we applied genome-wide transcript profiling to gain a global picture of the ways in which a large proportion of genes are expressed in the immature ear tissues of a series of 16 maize hybrids that vary in their degree of heterosis. Key observations include: (1) the proportion of allelic additively expressed genes is positively associated with hybrid yield and heterosis; (2) the proportion of genes that exhibit a bias towards the expression level of the paternal parent is negatively correlated with hybrid yield and heterosis; and (3) there is no correlation between the over- or under-expression of specific genes in maize hybrids with either yield or heterosis. The relationship of the expression patterns with hybrid performance is substantiated by analysis of a genetically improved modern hybrid (Pioneer hybrid 3394) versus a less improved older hybrid (Pioneer hybrid 3306) grown at different levels of plant density stress. The proportion of allelic additively expressed genes is positively associated with the modern high yielding hybrid, heterosis and high yielding environments, whereas the converse is true for the paternally biased gene expression. The dynamic changes of gene expression in hybrids responding to genotype and environment may result from differential regulation of the two parental alleles. Our findings suggest that differential allele regulation may play an important role in hybrid yield or heterosis, and provide a new insight to the molecular understanding of the underlying mechanisms of heterosis.

Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5' ADH1 matrix attachment region.

Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix. Thus, they may define discrete gene-containing chromatin loops in vivo. We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants. Three MAR elements, two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. In callus, two of the MAR elements (Adh1 5' MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression. In transgenic plants, Adh1 5' MAR had the effect of localizing beta-glucuronidase expression to lateral root initiation sites. A possible model accounting for the function of Adh1 5' MAR is discussed.

Massively parallel signature sequencing (MPSS) as a tool for in-depth quantitative gene expression profiling in all organisms.

Massively parallel signature sequencing (MPSS) is one of the newest tools available for conducting in-depth expression profiling. MPSS is an open-ended platform that analyses the level of expression of virtually all genes in a sample by counting the number of individual mRNA molecules produced from each gene. There is no requirement that genes be identified and characterised prior to conducting an experiment. MPSS has a routine sensitivity at a level of a few molecules of mRNA per cell, and the datasets are in a digital format that simplifies the management and analysis of the data. Therefore, of the various microarray and non-microarray technologies currently available, MPSS provides many advantages for generating the type of complete datasets that will help to facilitate hypothesis-driven experiments in the era of digital biology.