Single-cell resolution of human airway epithelial cells exposed to bronchiolitis obliterans-associated chemicals.

Bronchiolitis obliterans (BO) is a fibrotic lung disease characterized by progressive luminal narrowing and obliteration of the small airways. In the nontransplant population, inhalation exposure to certain chemicals is associated with BO; however, the mechanisms contributing to disease induction remain poorly understood. This study's objective was to use single-cell RNA sequencing for the identification of transcriptomic signatures common to primary human airway epithelial cells after chemical exposure to BO-associated chemicals-diacetyl or nitrogen mustard-to help explain BO induction. Primary airway epithelial cells were cultured at air-liquid interface and exposed to diacetyl, nitrogen mustard, or control vapors. Cultures were dissociated and sequenced for single-cell RNA. Differential gene expression and functional pathway analyses were compared across exposures. In total, 75,663 single cells were captured and sequenced from all exposure conditions. Unbiased clustering identified 11 discrete phenotypes, including 5 basal, 2 ciliated, and 2 secretory cell clusters. With chemical exposure, the proportion of cells assigned to keratin 5+ basal cells decreased, whereas the proportion of cells aligned to secretory cell clusters increased compared with control exposures. Functional pathway analysis identified interferon signaling and antigen processing/presentation as pathways commonly upregulated after diacetyl or nitrogen mustard exposure in a ciliated cell cluster. Conversely, the response of airway basal cells differed significantly with upregulation of the unfolded protein response in diacetyl-exposed basal cells, not seen in nitrogen mustard-exposed cultures. These new insights provide early identification of airway epithelial signatures common to BO-associated chemical exposures.NEW & NOTEWORTHY Bronchiolitis obliterans (BO) is a devastating fibrotic lung disease of the small airways, or bronchioles. This original manuscript uses single-cell RNA sequencing for identifying common signatures of chemically exposed airway epithelial cells in BO induction. Chemical exposure reduced the proportion of keratin 5+ basal cells while increasing the proportion of keratin 4+ suprabasal cells. Functional pathways contributory to these shifts differed significantly across exposures. These new results highlight similarities and differences in BO induction across exposures.

Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner.

Plants establish mutualistic associations with beneficial microbes while deploying the immune system to defend against pathogenic ones. Little is known about the interplay between mutualism and immunity and the mediator molecules enabling such crosstalk. Here, we show that plants respond differentially to a volatile bacterial compound through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. We found that exposure of Arabidopsis thaliana to a known plant growth-promoting rhizobacterium can unexpectedly have either beneficial or deleterious effects to plants. The beneficial-to-deleterious transition is dependent on availability of phosphate to the plants and is mediated by diacetyl, a bacterial volatile compound. Under phosphate-sufficient conditions, diacetyl partially suppresses plant production of reactive oxygen species (ROS) and enhances symbiont colonization without compromising disease resistance. Under phosphate-deficient conditions, diacetyl enhances phytohormone-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Therefore, diacetyl affects the type of relation between plant hosts and certain rhizobacteria in a way that depends on the plant's phosphate-starvation response system and phytohormone-mediated immunity.

Combined assessment of lysine and N-acetyl cadaverine levels assist as a potential biomarker of the smoker periodontitis.

Periodontitis is an inflammatory condition of supporting structures of teeth leading to attachment and bone loss. Cigarette smoking is the single most important and modifiable risk factor with 5 to 20-fold susceptibility for periodontal diseases. Reverse smoking is a peculiar habit of smoking where the lit end is kept inside the mouth, which is predominant in the northern coastal districts of Andhra Pradesh. Polyamines are biologically active amines involved in tissue regeneration and modulation of inflammation. The study aimed to evaluate polyamines and check their utility as a marker in detection of periodontitis among different groups. Total polyamine levels showed significant increase in reverse smokers with periodontitis when compared to the other groups. Qualitative analysis by thin layer chromatography showed three polyamine bands with varying intensity among the different groups. Mass spectrometric and NMR analyses of the three bands identified them as N1, N8-diacetyl spermidine, N-acetyl cadaverine and lysine. Most significantly elevated levels of lysine was observed in the smoker and reverse smoker periodontitis groups when compared to healthy and non-smoker periodontitis groups. The significantly elevated levels of N-acetyl cadaverine could be responsible for the more destruction of periodontium in the reverse smoker group. Antioxidant potential decreased significantly in different smoker periodontitis groups. The present study suggests that the quantitative analysis of salivary polyamines, lysine and N-acetyl cadaverine can aid as an easy noninvasive diagnostic method for assessing the periodontal status, especially in smokers.

IL-17A neutralization fails to attenuate airway remodeling and potentiates a proinflammatory lung microenvironment in diacetyl-exposed rats.

Bronchiolitis obliterans (BO) is a devastating lung disease that can develop following inhalation exposure to certain chemicals. Diacetyl (DA) is one chemical commonly associated with BO development when inhaled at occupational levels. Previous studies in rats have shown that repetitive DA vapor exposures increased lung CD4+CD25+ T cells and bronchoalveolar (BAL) interleukin-17A (IL-17A) concentrations concurrent with the development of airway remodeling. We hypothesized that IL-17A neutralization would attenuate the severity of airway remodeling after repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor or filtered air (RA) for 6 h/day × 5 days and monitored for 2 wk postexposure. Treatment with IL-17A neutralization (αIL-17A) or IgG (control) began immediately following exposures and continued twice weekly until study's end. Lungs were harvested for histology, flow cytometry, and BAL analyses. Survival, oxygen saturations, and percent weight change decreased significantly in DA-exposed versus RA-exposed rats, but did not differ significantly between DA + αIL-17A versus DA + IgG. Similarly, the number nor severity of airway lesions did not differ significantly between DA + αIL-17A versus DA + IgG rats despite the percentage of lung regulatory T cells increasing with decreased BAL IL-17A concentrations. Ashcroft scoring of the distal lung parenchyma suggested worse parenchymal remodeling in DA + αIL-17A versus DA + IgG rats with increased expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and nuclear factor-kappa B (NF-κB). Collectively, IL-17A neutralization in DA-exposed rats failed to attenuate airway remodeling with increased expression of pro-inflammatory cytokines TNF-α, IL-1ß, and NF-κB.NEW & NOTEWORTHY Interleukin-17A (IL-17A) neutralization has shown benefit previously in preclinical models of transplant-associated bronchiolitis obliterans (BO), yet it remains unknown whether IL-17A neutralization has similar benefit for other forms of BO. Here, IL-17A neutralization fails to prevent severe airway remodeling in rats exposed repetitively to the flavoring chemical diacetyl, and instead, promotes a proinflammatory microenvironment with increased expression of TNF-α, IL-1ß, and NF-κB within the lung.

Enzymes and pathways in microbial production of 2,3-butanediol and 3-acetoin isomers.

2,3-Butanediol (BD) and acetoin (AC) are products of the non-oxidative metabolism of microorganisms, presenting industrial importance due to their wide range of applications and high market value. Their optical isomers have particular applications, justifying the efforts on the selective bioproduction. Each microorganism produces different isomer mixtures, as a consequence of having different butanediol dehydrogenase (BDH) enzymes. However, the whole scene of the isomer bioproduction, considering the several enzymes and conditions, has not been completely elucidated. Here we show the BDH classification as R, S or meso by bioinformatics analysis uncovering the details of the isomers production. The BDH was compared to diacetyl reductases (DAR) and the new enoyl reductases (ER). We observed that R-BDH is the most singular BDH, while meso and S-BDHs are similar and may be better distinguished through their stereo-selective triad. DAR and ER showed distinct stereo-triads from those described for BDHs, agreeing with kinetic data from the literature and our phylogenetic analysis. The ER family probably has meso-BDH like activity as already demonstrated for a single sequence from this group. These results are of great relevance, as they organize BD producing enzymes, to our known, never shown before in the literature. This review also brings attention to nontraditional enzymes/pathways that can be involved with BD/AC synthesis, as well as oxygen conditions that may lead to the differential production of their isomers. Together, this information can provide helpful orientation for future studies in the field of BD/AC biological production, thus contributing to achieve their production on an industrial scale.

Two Fast GC-MS Methods for the Measurement of Nicotine, Propylene Glycol, Vegetable Glycol, Ethylmaltol, Diacetyl, and Acetylpropionyl in Refill Liquids for E-Cigarettes.

The use of e-cigarettes (ECs) has become increasingly popular worldwide, even though scientific results have not established their safety. Diacetyl (DA) and acetylpropionyl (AP), which can be present in ECs, are linked with lung diseases. Ethyl maltol (EM)-the most commonly used flavoring agent-can be present in toxic concentrations. Until now, there is no methodology for the determination of nicotine, propylene glycol (PG), vegetable glycerin (VG), EM, DA, and acetylpropionyl in e-liquids that can be used as a quality control procedure. Herein, gas chromatography coupled with mass spectrometry (GC-MS) was applied for the development of analytical methodologies for these substances. Two GC-MS methodologies were developed and fully validated, fulfilling the standards for the integration in a routine quality control procedure by manufacturers. As proof of applicability, the methodology was applied for the analysis of several e-liquids. Differences were observed between the labeled and the experimental levels of PG, VG, and nicotine. Three samples contained EM at higher concentrations compared to the other samples, while only one contained DA. These validated methodologies can be used for the quality control analysis of EC liquid samples regarding nicotine, PG, and VG amounts, as well as for the measurement of the EM.

Molecular and Electronic Structures of Macrocyclic Compounds Formed at Template Synthesis in the M(II)-Thiocarbohydrazide-Diacetyl Triple Systems: A Quantum-Chemical Analysis by DFT Methods.

Using density functional theory (DFT) B3PW91/TZVP, M06/TZVP, and OPBE/TZVP chemistry models and the Gaussian09 program, a quantum-chemical calculation of geometric and thermodynamic parameters of Ni(II), Cu(II), and Zn(II) macrotetracyclic chelates, with (NNNN)-coordination of ligand donor centers arising during template synthesis between the indicated ions of 3d elements, thiocarbohydrazide H2N-HN-C(=S)-NH-NH2 and diacetyl Me-C(=O)-C(=O)-Me, in gelatin-immobilized matrix implants was performed. The key bond lengths and bond angles in these coordination compounds are provided, and it is noted that in all these complexes the MN4 chelate sites, the grouping of N4 atoms bonded to the M atom, and the five-membered and six-membered metal chelate rings are practically coplanar. NBO analysis of these compounds was carried out, on the basis of which it was shown that all these complexes, in full accordance with theoretical expectations, are low-spin complexes. The standard thermodynamic characteristics of the template reactions for the formation of the above complexes are also presented. Good agreement between the data obtained using the above DFT levels is noted.

[Two cases of airway dysfunction related to diacetyl exposure].

Occupational exposure to diacetyl can lead to bronchiolitis obliterans. In this paper, two patients with severe obstructive ventilation disorder who were exposed to diacetyl at a fragrance and flavours factory were analyzed. The clinical manifestations were cough and shortness of breath. One of them showed Mosaic shadows and uneven perfusion in both lungs on CT, while the other was normal. Field investigation found that 4 of the 8 workers in the factory were found to have obstructive ventilation disorder, and 2 had small airway dysfunction. This paper summarizes the diagnostic process of patients in order to improve the understanding of airway dysfunction caused by occupational exposure to diacetyl and promote the development of relevant standards.

Diacetyl inhalation impairs airway epithelial repair in mice infected with influenza A virus.

Bronchiolitis obliterans (BO) is a debilitating disease of the small airways that can develop following exposure to toxic chemicals as well as respiratory tract infections. BO development is strongly associated with diacetyl (DA) inhalation exposures at occupationally relevant concentrations or severe influenza A viral (IAV) infections. However, it remains unclear whether lower dose exposures or more mild IAV infections can result in similar pathology. In the current work, we combined these two common environmental exposures, DA and IAV, to test whether shorter DA exposures followed by sublethal IAV infection would result in similar airways disease. Adult mice exposed to DA vapors 1 h/day for 5 consecutive days followed by infection with the airway-tropic IAV H3N2 (HKx31) resulted in increased mortality, increased bronchoalveolar lavage (BAL) neutrophil percentage, mixed obstruction and restriction by lung function, and subsequent airway remodeling. Exposure to DA or IAV alone failed to result in significant pathology, whereas mice exposed to DA + IAV showed increased α-smooth muscle actin (αSMA) and epithelial cells coexpressing the basal cell marker keratin 5 (KRT5) with the club cell marker SCGB1A1. To test whether DA exposure impairs epithelial repair after IAV infection, mice were infected first with IAV and then exposed to DA during airway epithelial repair. Mice exposed to IAV + DA developed similar airway remodeling with increased subepithelial αSMA and epithelial cells coexpressing KRT5 and SCGB1A1. Our findings reveal an underappreciated concept that common environmental insults while seemingly harmless by themselves can have catastrophic implications on lung function and long-term respiratory health when combined.

Effect of alsD deletion and overexpression of nox and alsS on diacetyl and acetoin production by Lacticaseibacillus casei during milk fermentation.

Diacetyl and acetoin are key aroma components of fermented milk but are produced in low concentrations by starter cultures. In this study, we expressed NADH oxidase, acetolactate synthase, and inactivated acetolactate decarboxylase in Lacticaseibacillus casei TCS to generate recombinant L. casei strains, and investigated the effects of the genes encoding these enzymes on diacetyl and acetoin production during milk fermentation. In the single-gene recombinant strains tested, diacetyl concentrations were highest in milk fermented by L. casei TCSI-nox (nox gene overexpressed, 3.68 mg/kg), whereas acetoin concentrations were highest in milk fermented by L. casei TCS-ΔalsD (alsD gene deleted, 32.94 mg/kg). Moreover, diacetyl and acetoin concentrations were higher in the inducible strains than in the corresponding constitutive strains (e.g., TCSI-nox vs. TCSC-nox, and TCSI-ΔalsD-nox vs. TCSC-ΔalsD-nox). This phenomenon was also reflected in the protein expression levels and enzyme activities. In the double-gene recombinant strains tested, the highest concentrations of diacetyl and acetoin were produced by L. casei TCSI-ΔalsD-nox (nox overexpressed and alsD deleted, 4.66 mg/kg, 69.62 mg/kg, respectively). The triple-gene recombinant L. casei TCS-ΔalsD-nox-alsS produced the highest concentrations of diacetyl and acetoin, which were 2.38 and 11.19 times, respectively, the concentrations produced by the original strain. These results show that the nox, alsS, and alsD genes make key contributions to the biosynthesis of diacetyl and acetoin by L. casei. The modification of multiple genes had a synergistic effect, leading to greatly increased synthesis of diacetyl and acetoin by L. casei during its fermentation of milk.

Silymarin constrains diacetyl-prompted oxidative stress and neuroinflammation in rats: involvements of Dyn/GDNF and MAPK signaling pathway.

Neuroinflammation, a major component of many CNS disorders, has been suggested to be associated with diacetyl (DA) exposure. DA is commonly used as a food flavoring additive and condiment. Lately, silymarin (Sily) has shown protective and therapeutic effects on neuronal inflammation. The study aimed to explore the role of Sily in protecting and/or treating DA-induced neuroinflammation. Neuroinflammation was induced in rats by administering DA (25 mg/kg) orally. Results revealed that Sily (50 mg/kg) obviously maintained cognitive and behavioral functions, alleviated brain antioxidant status, and inhibited microglial activation. Sily enhanced IL-10, GDNF and Dyn levels, reduced IFN-γ, TNFα, and IL-1ß levels, and down-regulated the MAPK pathway. Immunohistochemical investigation of EGFR and GFAP declared that Sily could conserve neurons from inflammatory damage. However, with continuing DA exposure during Sily treatment, oxidative stress and neuroinflammation were less mitigated. These findings point to a novel mechanism involving the Dyn/GDNF and MAPK pathway through which Sily might prevent and treat DA-induced neuroinflammation.

Bacterial diacetyl suppresses abiotic stress-induced senescence in Arabidopsis.

Premature plant senescence induced by abiotic stresses is a major cause of agricultural losses worldwide. Tools for suppressing stress-induced plant senescence are limited. Here, we report that diacetyl, a natural compound emitted by the plant-beneficial bacterium Bacillus amyloliquefaciens, suppresses abscisic acid -mediated foliar senescence in Arabidopsis thaliana under various abiotic stress conditions. Our results establish diacetyl as an effective protector against stress-induced plant senescence and reveal a molecular mechanism for bacteria-enhanced plant stress resistance.

[Quality markers of Zingiberis Rhizoma Carbonisata before and after processing].

Based on the previous research results of our group and literature research, the chemical components, mechanisms, pharmacodynamics, and pharmacokinetics of Zingiberis Rhizoma Carbonisata were summarized to determine the quality markers(Q-markers) of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma. Our research group has clarified the differential components of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma, the meridian-warming hemostatic effect of Zingiberis Rhizoma Carbonisata, the related targets and pathways of the effect, the endogenous biomarkers of Zingiberis Rhizoma Carbonisata, and the hemodynamic processes of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma. Moreover, based on high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry(HPLC-DAD-ESIMS), a method for determining the content of Q-mar-kers was established. In conclusion, the study finally determined that gingerone, 6-shogaol, and diacetyl-6-gingerol were the Q-mar-kers of Zingiberis Rhizoma Carbonisata decoction pieces, and 6-gingerol, 8-gingerol, and 10-gingerol were Q-markers of Zingiberis Rhizoma decoction pieces. The result is expected to provide a reference for the establishment of quality standards for Zingiberis Rhizoma Carbonisata decoction pieces and Zingiberis Rhizoma decoction pieces.

An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity.

Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.

Repetitive diacetyl vapor exposure promotes ubiquitin proteasome stress and precedes bronchiolitis obliterans pathology.

Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant, following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha-diketone known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6 h per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14 days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein ubiquitin-C accumulated within DA-exposed rat lung homogenates and localized primarily to the airway epithelium, the primary site of BO development. Lung proteasome activity increased concurrently with ubiquitin-C expression after DA exposure, supportive of significant proteasome stress. In primary human airway cultures, global proteomics identified 519 significantly modified proteins in DA-exposed samples relative to controls with common pathways of the ubiquitin proteasome system, endosomal reticulum transport, and response to unfolded protein pathways being upregulated and cell-cell adhesion and oxidation-reduction pathways being downregulated. Collectively, these two models suggest that diacetyl inhalation exposure causes abundant protein damage and subsequent ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.

Diacetyl exposure disrupts iron homeostasis in animals and cells.

OBJECTIVE: Several mechanisms have been proposed for the biological effect of diacetyl. We tested the postulate that animal and cell exposures to diacetyl are associated with a disruption in iron homeostasis. MATERIALS AND METHODS: Male, Sprague-Dawley rats were intratracheally-instilled with either distilled water or diacetyl. Seven days after treatment, animals were euthanized and the lungs removed, fixed, and embedded. Sections were cut and stained for iron, collagen, and ferritin. Human epithelial (BEAS-2B) and monocytic (THP-1) cells were exposed in vitro to ferric ammonium citrate (FAC), diacetyl, and both FAC and diacetyl. Cell non-heme iron concentrations and ferritin levels were quantified using inductively coupled plasma optical emission spectroscopy and an immunoassay respectively. RESULTS: After exposure of animals to diacetyl, there were airway polypoid lesions which stained positively for both iron and the intracellular storage protein ferritin. Trichrome stain showed a deposition of collagen immediately adjacent to accumulated metal following diacetyl exposure. In in vitro cell exposures, FAC increased non-heme iron concentration but co-incubations of FAC and diacetyl elevated levels to significantly greater values. Levels of ferritin were increased with exposures of BEAS-2B and THP-1 cells to FAC but were similarly greater after co-exposure with FAC and diacetyl. CONCLUSIONS: Results of animal and cell studies support a disruption of iron homeostasis by diacetyl. It is proposed that, following internalization, diacetyl complexes intracellular sources of iron. The cell recognizes a loss of its requisite iron to diacetyl and imports greater concentrations of the metal.

[Advances in flavoring-related bronchiolitis obliterans].

Occupational exposure to the diacetyl flavoring may cause bronchiolitis obliterans (BO) in workers. The first case of flavoring-related bronchiolitis obliterans was reported in 2002. Since then, similar cases have been identified among workers in various production industries in some countries and regions. At present, there are no cases reported in China. In order to improve the awareness of the disease and promote the prevention work, this article reviews the research progress of case recognition, risk factors, clinical manifestation and possible pathogenesis.

Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate.

BACKGROUND: Diacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl. RESULTS: By expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate. CONCLUSIONS: An efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.

Flavorings-Related Lung Disease: A Brief Review and New Mechanistic Data.

Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile α-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon α-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate α-dicarbonyl compounds in airway injury and flavorings-related lung disease.

Influence of sugars and pH on the citrate metabolism of different lactic acid bacteria strains in a synthetic wine matrix.

AIMS: This study investigated the influence of sugars (glucose and fructose) and pH on the gene expression of citE (citrate lyase ß-subunit) and the subsequent formation of metabolites associated with citrate metabolism. METHODS AND RESULTS: Different levels of glucose (2·5, 50 and 115 g l-1 ), fructose (2·5, 50 and 115 g l-1 ) and pH (3·0, 3·5, 4·0 and 5·0) were evaluated for their effect on citE expression in four different lactic acid bacteria strains. Two Oenococcus oeni strains and two Lactobacillus plantarum strains were used, of which one strain of each species screened positive for the citE gene. Among the factors tested, fructose had the biggest influence on the relative expression of citE in O. oeni. In addition, the citrate-positive strains produced high concentrations of diacetyl and acetoin. CONCLUSIONS: This study gives an overview of how sugar, pH and different lactic acid bacteria strains influence citE gene expression and the formation of metabolites associated with citrate metabolism closely linked to malolactic fermentation (MLF). SIGNIFICANCE AND IMPACT OF THE STUDY: These results can be used to make informed decisions regarding MLF when aiming to create a wine with a buttery aroma or not.