Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

A high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) as an elemental specific detector was used for the speciation analysis of arsenic compounds in urine and serum samples from Vietnam. Five arsenic species including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB) were studied. A gradient elution of ammonium carbonate ((NH4)2CO3), ethylenediaminetetraacetic acid disodium salt (Na2EDTA), and methanol at pH 9.0 utilizing Hamilton PRP-X100 strong anion-exchange column allowed the chromatographic separation of five arsenic species. In this study, urine and serum samples were prepared by dilution in solvent and protein precipitation by trichloroacetic acid, respectively. The extraction efficiency was greater than 91% for urine matrix, and recoveries from spiked samples were in the range of 94–139% for the arsenic species in human serum. The method limit of detection (MDL) and limit of quantification (MQL), which were calculated by signal to noise ratio, were found to be 0.3–1.5 and 1.0–5.0 ng·mL−1, respectively. The concentration of arsenic species in 17 pairs of urine and serum samples from Vietnam was also quantified and evaluated. The major species of arsenic in the urine and serum samples were AsB and DMA.

Advances in omics technologies now permit generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells, and high-resolution determination of gene regulatory features including 3-dimensional chromatin interactions. Using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anti-cancer drugs. We identified not only new clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes but also rampant gene duplication including paralogs of MIA pathway genes. Clustering was not limited to the linear genome and through chromatin interaction data, MIA pathway genes were shown to be present within the same topologically associated domain, permitting identification of a secologanin transporter. Single cell RNA-sequencing revealed exquisite and sequential cell-type specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a newly developed single cell metabolomics approach, permitted identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. Last, we revealed cell-type specific expression in the root MIA pathway that is conferred in part by neo- and sub-functionalization of paralogous MIA pathway genes. This study highlights how a suite of omic approaches, including single cell gene expression and metabolomics, can efficiently link sequence with function in complex, specialized metabolic pathways of plants.

Acidities of lipophilic compounds, such as various ligands or catalysts, in systems consisting of an aqueous phase at equilibrium with a water-immiscible phase (lipid bilayers, phase transfer catalysis, sensor membranes, to name just few) are typically approximated by the aqueous pK a values. Our research shows that such approximations can lead to seriously biased estimations of the acidities as the bulk of solvated H+ ions reside in the aqueous phase, while the lipophilic speciesboth neutral acid and anionpredominantly reside in the organic phase. Therefore, the use of aqueous pK a in such situations is not justified. In this work, we provide a more accurate description of the acidities of acids in such systems by applying the biphasic pK a concept. Biphasic pK a values (pK a ow values) of 35 acids of various structures and chemical properties were determined in a 1-octanol:water system. We provide detailed descriptions of the UV–vis and NMR measurement methods. The directly obtained (apparent) pK a ow values depend on concentration. Concentration-independent values were obtained by extrapolating the apparent values to zero concentration using a Debye–Hückel model.