Accurate selected ion flow tube mass spectrometry quantification of ethylene oxide contamination in the presence of acetaldehyde.

In September 2020, traces of ethylene oxide (a toxic substance used as a pesticide in developing countries but banned for use on food items within the European Union) were found in foodstuffs containing ingredients derived from imported sesame seed products. Vast numbers of foodstuffs were recalled across Europe due to this contamination, leading to expensive market losses and extensive trace exposure of ethylene oxide to consumers. Therefore, a rapid analysis method is needed to ensure food safety by high-throughput screening for ethylene oxide contamination.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds.

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of and Three Candidate Species From China and Japan.

JAD2, the only cultured representative of the order , is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for JAD2 and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of JAD2 and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing " Thermoflexus japonica," " Thermoflexus tengchongensis," and " Thermoflexus sinensis.

Exometabolomics and MSI: deconstructing how cells interact to transform their small molecule environment.

Metabolism is at the heart of many biotechnologies from biofuels to medical diagnostics. Metabolomic methods that provide glimpses into cellular metabolism have rapidly developed into a critical component of the biotechnological development process. Most metabolomics methods have focused on what is happening inside the cell.

Benchtop isolation and characterization of functional exosomes by sequential filtration.

Early and minimally invasive detection of malignant events or other pathologies is of utmost importance in the pursuit of improved patient care and outcomes. Recent evidence indicates that exosomes and extracellular vesicles in serum and body fluids can contain nucleic acid, protein, and other biomarkers. Accordingly, there is great interest in applying these clinically as prognostic, predictive, pharmacodynamic, and early detection indicators.

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells.

L-asparaginase (L-ASP) is a therapeutic enzyme used clinically for the treatment of childhood acute lymphoblastic leukemia. L-ASP's anticancer activity is believed to be associated primarily with depletion of asparagine, but secondary glutaminase activity has also been implicated in its anticancer mechanism of action. To investigate the effects of L-ASP on amino acid metabolism, we have developed an LC-MS/MS metabolomics platform for high-throughput quantitation of 29 metabolites, including all 20 proteinogenic amino acids, 6 metabolically related amino acid derivatives (ornithine, citrulline, sarcosine, taurine, hypotaurine, and cystine), and 3 polyamines (putrescince, spermidine, and spermine) in adherent cultured cells.

An artifact in LC-MS/MS measurement of glutamine and glutamic acid: in-source cyclization to pyroglutamic acid.

Advances in metabolomics, particularly for research on cancer, have increased the demand for accurate, highly sensitive methods for measuring glutamine (Gln) and glutamic acid (Glu) in cell cultures and other biological samples. N-terminal Gln and Glu residues in proteins or peptides have been reported to cyclize to pyroglutamic acid (pGlu) during liquid chromatography (LC)-mass spectrometry (MS) analysis, but cyclization of free Gln and Glu to free pGlu during LC-MS analysis has not been well-characterized. Using an LC-MS/MS protocol that we developed to separate Gln, Glu, and pGlu, we found that free Gln and Glu cyclize to pGlu in the electrospray ionization source, revealing a previously uncharacterized artifact in metabolomic studies.

Measurement of DNA concentration as a normalization strategy for metabolomic data from adherent cell lines.

Metabolomics is a rapidly advancing field, and much of our understanding of the subject has come from research on cell lines. However, the results and interpretation of such studies depend on appropriate normalization of the data; ineffective or poorly chosen normalization methods can lead to frankly erroneous conclusions. That is a recurrent challenge because robust, reliable methods for normalization of data from cells have not been established.

The membrane-binding domain of an amphitropic enzyme suppresses catalysis by contact with an amphipathic helix flanking its active site.

CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in the synthesis of phosphatidylcholine, is activated by binding membranes using a lipid-induced amphipathic helix (domain M). Domain M functions to silence catalysis when CCT is not membrane engaged. The silencing mechanism is unknown.

Assembly of Ebola virus matrix protein VP40 is regulated by latch-like properties of N and C terminal tails.

The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.

Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation.

Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood.

Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin.

The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA.

Conserved interaction between transferrin and transferrin-binding proteins from porcine pathogens.

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf.

Proteomics by mass spectrometry--go big or go home?

Mass spectrometry is an important technology for mapping composition and flux in whole proteomes. Over the last 5 years in particular, impressive gains in the depth of proteome coverage have been realized, particularly for model organisms. This review will provide an update on advancements in the key analytical techniques, methods and informatics directed towards whole proteome analysis by mass spectrometry.

Structural variations within the transferrin binding site on transferrin-binding protein B, TbpB.

Pathogenic bacteria acquire the essential element iron through specialized uptake pathways that are necessary in the iron-limiting environments of the host. Members of the Gram-negative Neisseriaceae and Pasteurellaceae families have adapted to acquire iron from the host iron binding glycoprotein, transferrin (Tf), through a receptor complex comprised of transferring-binding protein (Tbp) A and B. Because of the critical role they play in the host, these surface-exposed proteins are invariably present in clinical isolates and thus are considered prime vaccine targets.