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Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the networks regulatory outputs and a decrease in fitness. We subjected these flies to EMS mutagenesis, followed by experimental evolution. After only 8-15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level.

Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations. Electrospray post-ionization can be used to circumvent many sample cleanup and desalting steps. SESI and IR-MALDESI are two examples of ionization methods that employ electrospray post-ionization at atmospheric pressure and temperature. By coupling the two techniques and doping the electrospray solvent with silver ions, olefinic terpenes of different classes and varying degrees of volatility were directly analyzed from a biological buffer system with no sample workup steps. [ABSTRACT FROM AUTHOR]

In the field of mass spectrometry imaging, typical experiments involve ionization directly from complex samples with no pre-ionization separation, relying on high resolving power mass analyzers to separate ions of interest. When an ion trapping step is involved in the analysis, the dynamic range of the analysis may be limited by the capacity of the ion trap, which is easily exceeded. To minimize collection of undesired ambient species while maximizing collection of analyte signal, accurate timing between ion generation and collection is a requirement. Here, a method for achieving synchronicity between infrared laser ablation and ion collection on a Q Exactive Plus mass spectrometer is described and demonstrated through measurement of ion accumulation at fixed time points following a laser ablation event with electrospray post-ionization of ablated material. In a model imaging experiment using infrared matrix-assisted laser desorption electrospray ionization, fixing the injection time at the minimum duration required to capture all ions generated by the last laser pulse in a sequence is shown to maximize target ion abundances. Using optimized timing is shown to yield a doubling or better of useful signal compared to previously used parameters. [ABSTRACT FROM AUTHOR]

Rationale: High-throughput screening (HTS) is a critical step in the drug discovery process. However, most mass spectrometry (MS)-based HTS methods require sample cleanup steps prior to analysis. In this work we present the utility of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for monitoring an enzymatic reaction directly from a biological buffer system with no sample cleanup and at high throughput. Methods: IR-MALDESI was used to directly analyze reaction mixtures from a well plate at different time points after reaction initiation. The percent conversion of precursors to products was used to screen the enzyme activity. The reaction was performed with two different concentrations of precursors and enzyme in order to assess the dynamic range of the assay. Eventually, a pseudo-HTS study was designed to investigate the utility of IR-MALDESI screening enzyme activity in a high-throughput manner. Results: IR-MALDESI was able to readily monitor the activity of IDH1 over time at two different concentrations of precursors and enzyme. The calculated Z-factors of 0.65 and 0.41 confirmed the suitability of the developed method for screening enzyme activity in HTS manner. Finally, in a single-blind pseudo-HTS analysis IR-MALDESI was able to correctly predict the identity of all samples, where 8/10 samples were identified with high confidence and the other two samples with lower confidence. Conclusions: The enzymatic activity of IDH1 was screened by directly analyzing the reaction content from the buffer in well plates with no sample cleanup steps. This proof-of-concept study demonstrates the robustness of IR-MALDESI for direct analysis of enzymatic reactions from biological buffers with no sample cleanup and its immense potential for HTS applications. [ABSTRACT FROM AUTHOR]

Metabolism has emerged as a key factor in homeostasis and disease including cancer. Yet, little is known about the heterogeneity of metabolic activity of cancer cells due to the lack of tools to directly probe it. Here, we present a novel method, 13 C-SpaceM for spatial single-cell isotope tracing of glucose-dependent de novo lipogenesis. The method combines imaging mass spectrometry for spatially-resolved detection of 13 C 6 -glucose-derived 13 C label incorporated into esterified fatty acids with microscopy and computational methods for data integration and analysis. We validated 13 C-SpaceM on a spatially-heterogeneous normoxia-hypoxia model of liver cancer cells. Investigating cultured cells, we revealed single-cell heterogeneity of lipogenic acetyl-CoA pool labelling degree upon ACLY knockdown that is hidden in the bulk analysis and its effect on synthesis of individual fatty acids. Next, we adapted 13 C-SpaceM to analyze tissue sections of mice harboring isocitrate dehydrogenase (IDH)-mutant gliomas. We found a strong induction of de novo fatty acid synthesis in the tumor tissue compared to the surrounding brain. Comparison of fatty acid isotopologue patterns revealed elevated uptake of mono-unsaturated and essential fatty acids in the tumor. Furthermore, our analysis uncovered substantial spatial heterogeneity in the labelling of the lipogenic acetyl-CoA pool indicative of metabolic reprogramming during microenvironmental adaptation. Overall, 13 C-SpaceM enables novel ways for spatial probing of metabolic activity at the single cell level. Additionally, this methodology provides unprecedented insight into fatty acid uptake, synthesis and modification in normal and cancerous tissues., Competing Interests: Conflict of interest T.A. holds patents on imaging mass spectrometry and leads a startup on single-cell metabolomics incubated at the BioInnovation Institute. G.J.P. is a scientific advisory board member for Cambridge Isotope Laboratories and has a collaborative research agreement with Thermo Scientific.