Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is, therefore, of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD-dependent methanol dehydrogenase (Mdh) variants, the rate-limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross-talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion.Finally, we applied this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build-up while also reducing the burden of heterologous expression.This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy. pastoris convert methanol to formaldehyde using an FAD-linked alcohol oxidase (AOX) (Cregg, Madden, Barringer, Thill, & Stillman, 1989), and Gram-positive methylotrophs typified by Bacillus methanolicus perform the same conversion using an NAD-linked Mdh (Müller, Heggeset, Wendisch, Vorholt, & Brautaset, 2015).In all these organisms, formaldehyde acts as a branch point between further oxidization to CO 2 for energy conservation, and incorporation into biomass via the serine cycle, ribulose monophosphate (RuMP) pathway, or the xylulose-5-phosphate (Xu5P) pathway (Chistoserdova et al., 2009;Müller, Heggeset, et al., 2015;Yurimoto, Oku, & Sakai, 2011). These pathways are of growing interest in the field of metabolic engineering, where researchers seek to convert relatively cheap methanol feedstocks into higher value commodity chemicals with either native or "synthetic" methylotrophs (Kalyuzhnaya, Puri, & Lidstrom, 2015;Whitaker, Sandoval, Bennett, Fast, & Papoutsakis, 2015). Besides methylotrophs, formaldehyde is present at low levels in all organisms as a result of demethylation reactions (Kalasz, 2003). Because of its cytotoxicity, the intracellular formaldehyde concentration must be tightly controlled, which has led to the evolution of a variety of highly coordinated metabolic strategies for detoxifying formaldehyde (Yurimoto, Kato, & Sakai, 2005). The need to keep the concentration of formaldehyde low while supporting high flux places an even more stringent burden on methylotrop...

Human tissue samples commonly preserved as formalin-fixed paraffin-embedded (FFPE) tissues after diagnostic or surgical procedures in the clinic represent an invaluable source of clinical specimens for indepth characterization of signaling networks to assess therapeutic options. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. Additionally, pTyr analysis that may otherwise inform therapeutic interventions for patients has been limited by the requirement for large amounts of frozen tissue. Here we describe a method for highly sensitive, quantitative analysis of pTyr signaling networks, with hundreds of sites quantified from 1-2 10-µm sections of FFPE tissue specimens. A combination of optimized magnetic bead-based sample processing, optimized pTyr enrichment strategies, and TMT multiplexing enabled in depth coverage of pTyr signaling networks from small amounts of input material. Phosphotyrosine profiles of flash frozen and FFPE tissues derived from the same tumors suggested that FFPE tissues preserve pTyr signaling characteristics in patient-derived xenografts and archived clinical specimens. pTyr analysis of FFPE tissue sections from breast cancer tumors as well as lung cancer tumors highlighted patient-specific oncogenic driving kinases, indicating potential targeted therapies for each patient. These data suggest the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens.

Highlights d AAG-initiated base excision repair modulates susceptibility to NDMA-induced disease d Low AAG increases susceptibility to methylation-induced mutations and liver cancer d High AAG activity sensitizes animals to hepatotoxicity and lethality from NDMA d Integration of phenotypes over time reveals progression of NDMA-induced pathologies Authors

Background. Polycyclic aromatic hydrocarbons (PAHs) emitted from combustion sources are known to be mutagenic, with more potent species also being carcinogenic. Previous studies show that PAHs can undergo complex transformations both in the body and in the atmosphere, yet these transformation processes are generally investigated separately. Objectives. Drawing from the literature in atmospheric chemistry and toxicology, we highlight the parallel transformations of PAHs that occur in the atmosphere and the body and discuss implications for public health. We also examine key uncertainties related to the toxicity of atmospheric oxidation products of PAHs and explore critical areas for future research. Discussion. We focus on a key mode of toxicity for PAHs, in which metabolic processes (driven by cytochrome P450 enzymes), leads to the formation of oxidized PAHs that can damage DNA. Such species can also be formed abiotically in the atmosphere from natural oxidation processes, potentially augmenting PAH toxicity by skipping the necessary metabolic steps that activate their mutagenicity. Despite the large body of literature related to these two general pathways, the extent to which atmospheric oxidation affects a PAH’s overall toxicity remains highly uncertain. Combining knowledge and promoting collaboration across both fields can help identify key oxidation pathways and the resulting products that impact public health. Conclusions. Cross-disciplinary research, in which toxicology studies evaluate atmospheric oxidation products and their mixtures, and atmospheric measurements examine the formation of compounds that are known to be most toxic. Close collaboration between research communities can help narrow down which PAHs, and which PAH degradation products, should be targeted when assessing public health risks. https://doi.org/10.1289/EHP9984