Transcriptome analysis reveals limited toxic effects of the UV-filter benzophenone-3 (BP-3) on the hermatypic coral Acropora tenuis and its symbiotic dinoflagellates.

This study aimed to investigate the toxic and transcriptomic effects of the ultraviolet filter benzophenone-3 (BP-3) on Acropora tenuis and its symbiotic dinoflagellates while using acetone as a solvent. Seven-day exposure to 50 and 500 µg/L, which is higher than most BP-3 records from coastal waters, did not affect coral colour or dinoflagellate photosynthesis. Differentially expressed genes (DEGs) between seawater and solvent controls were <20 in both corals and dinoflagellates. Eleven coral DEGs were detected after treatment with 50 µg/L BP-3. Fourteen coral DEGs, including several fluorescent protein genes, were detected after treatment with 500 µg/L BP-3. In contrast, no dinoflagellate DEGs were detected in the BP-3 treatment group. These results suggest that the effects of 50-500 µg/L BP-3 on adult A. tenuis and its dinoflagellates are limited. Our experimental methods with lower acetone toxicity provide a basis for establishing standard ecotoxicity tests for corals.

Genome-wide characterization of circulating metabolic biomarkers.

Genome-wide association analyses using high-throughput metabolomics platforms have led to novel insights into the biology of human metabolism1-7. This detailed knowledge of the genetic determinants of systemic metabolism has been pivotal for uncovering how genetic pathways influence biological mechanisms and complex diseases8-11. Here we present a genome-wide association study for 233 circulating metabolic traits quantified by nuclear magnetic resonance spectroscopy in up to 136,016 participants from 33 cohorts. We identify more than 400 independent loci and assign probable causal genes at two-thirds of these using manual curation of plausible biological candidates. We highlight the importance of sample and participant characteristics that can have significant effects on genetic associations. We use detailed metabolic profiling of lipoprotein- and lipid-associated variants to better characterize how known lipid loci and novel loci affect lipoprotein metabolism at a granular level. We demonstrate the translational utility of comprehensively phenotyped molecular data, characterizing the metabolic associations of intrahepatic cholestasis of pregnancy. Finally, we observe substantial genetic pleiotropy for multiple metabolic pathways and illustrate the importance of careful instrument selection in Mendelian randomization analysis, revealing a putative causal relationship between acetone and hypertension. Our publicly available results provide a foundational resource for the community to examine the role of metabolism across diverse diseases.

Low levels of Cd2+ combined with procymidone may cause ovarian damage in mice via unfolded protein response.

As no study about the combined effect of low levels of Cd2+ with procymidone (PCM) on organs and organisms, we investigated their actions on mouse-ovary in vivo and in vitro. Four-week mice were treated with corn oil for the control group, corn oil + 0.0045 mg/L Cd2+ (CdCl2 was dissolved in ultrapure water and freely consumed by mice) for Cd2+ group, 50 mg/kg/d PCM (suspended in corn oil and administered orally to mice) for PCM group, and 50 mg/kg/d PCM + 0.0015 (0.0045 and 0.0135) mg/L Cd2+ for L+ (M+ and H+) PCM group for 21 days. For in vitro experiment, the cultured ovaries were treated with acetone for the control group, 0.1% acetone + 8.4 µg/L Cd2+ for the Cd2+ group, 0.63 mg/L PCM (dissolved in acetone) for the PCM-group, and 0.63 mg/L PCM + 2.8 (8.4 and 25.2) µg/L Cd2+ for L+ (M+ and H+) PCM group for 7 days. Mouse body weight in each treatment group, the weight and volume of ovaries in all PCM groups were lower than the control. Both in vivo and in vitro, all-stage follicle numbers were lower in M+PCM and H+PCM groups, whereas the atretic follicles and CASPASE3/8 were higher; meanwhile, lower estradiol and progesterone and higher unfolded protein response (UPR) members in all PCM groups. L+, M+, and H+PCM groups had further ovarian damage and stronger UPR than PCM groups, as did M+PCM groups over Cd2+ groups. It is hypothesized low-level PCM and Cd2+ may mutually promote each other's triggered UPR and exacerbate ovarian damage.

Methanol bioconversion into C3, C4, and C5 platform chemicals by the yeast Ogataea polymorpha.

BACKGROUND: One carbon (C1) molecules such as methanol have the potential to become sustainable feedstocks for biotechnological processes, as they can be derived from CO2 and green hydrogen, without the need for arable land. Therefore, we investigated the suitability of the methylotrophic yeast Ogataea polymorpha as a potential production organism for platform chemicals derived from methanol. We selected acetone, malate, and isoprene as industrially relevant products to demonstrate the production of compounds with 3, 4, or 5 carbon atoms, respectively. RESULTS: We successfully engineered O. polymorpha for the production of all three molecules and demonstrated their production using methanol as carbon source. We showed that the metabolism of O. polymorpha is well suited to produce malate as a product and demonstrated that the introduction of an efficient malate transporter is essential for malate production from methanol. Through optimization of the cultivation conditions in shake flasks, which included pH regulation and constant substrate feeding, we were able to achieve a maximum titer of 13 g/L malate with a production rate of 3.3 g/L/d using methanol as carbon source. We further demonstrated the production of acetone and isoprene as additional heterologous products in O. polymorpha, with maximum titers of 13.6 mg/L and 4.4 mg/L, respectively. CONCLUSION: These findings highlight how O. polymorpha has the potential to be applied as a versatile cell factory and contribute to the limited knowledge on how methylotrophic yeasts can be used for the production of low molecular weight biochemicals from methanol. Thus, this study can serve as a point of reference for future metabolic engineering in O. polymorpha and process optimization efforts to boost the production of platform chemicals from renewable C1 carbon sources.

A Quantitative Re-Assessment of Microencapsulation in (Pre-Treated) Yeast.

Most hydrophobes easily diffuse into yeast cells, where they experience reduced evaporation and protection from oxidation, thus allowing inherently biocompatible encapsulation processes. Despite a long-standing industrial interest, the effect of parameters such as how is yeast pre-treated (extraction with ethanol, plasmolysis with hypertonic NaCl, depletion to cell walls), the polarity of the hydrophobes and the process conditions are still not fully understood. Here, we have developed thorough analytical protocols to assess how the effects of the above on S. cerevisiae's morphology, permeability, and encapsulation efficiency, using three differently polar hydrophobes (linalool, 1,6-dihydrocarvone, limonene) and three separate processes (hydrophobes as pure 'oils', water dispersions, or acetone solutions). The harsher the pre-treatment (depleted > plasmolyzed/extracted > untreated cells), the easier the diffusion into yeast became, and the lower both encapsulation efficiency and protection from evaporation, possibly due to denaturation/removal of lipid-associated (membrane) proteins. More hydrophobic terpenes performed worst in encapsulation as pure 'oils' or in water dispersion, but much less of a difference existed in acetone. This indicates the specific advantage of solvents/dispersants for 'difficult' compounds, which was confirmed by principal component analysis; furthering this concept, we have used combinations of hydrophobes (e.g., linalool and α-tocopherol), with one acting as solvent/enhancer for the other. Our results thus indicate advantages in using untreated yeast and-if necessary-processes based on solvents/secondary hydrophobes.

Homologous acetone carboxylases select Fe(II) or Mn(II) as the catalytic cofactor.

Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

Model construction and theoretical evaluation of the performance improvement of acetone-butanol-ethanol extractive fermentation by adding surfactant.

Severe butanol toxicity to the metabolism of solventogenic clostridia significantly impede the application of fermentative butanol as a biofuel. Liquid-liquid extraction is an efficient method to reduce the butanol toxicity by in-situ removing it in the extractant phase. Butanol mass transfer into extractant phase in static acetone-butanol-ethanol (ABE) extractive fermentation with biodiesel as the extractant could be enhanced by adding a tiny amount of surfactant such as tween-80. In the case of corn-based ABE extractive fermentation by Clostridium acetobutylicum ATCC 824 using biodiesel originated from waste cooking oil as extractant, addition of 0.14% (w/v) tween-80 could increase butanol production in biodiesel and total solvents production by 21% and 17%, respectively, compared to those of control under non-surfactant existence. Furthermore, a mathematical model was developed to elucidate the mechanism of enhanced ABE extractive fermentation performance. The results indicated that the mass transfer improvement was obtained by effectively altering the physical properties of the self-generated bubbles during ABE extractive fermentation, such as reducing bubble size and extending its retention time in extractant phase, etc. Overall, this study provided an efficient approach for enhancing biobutanol production by integration of bioprocess optimization and model interpretation.

Transmission electron microscopy assessment of a novel method for isolating pure exosomes from serum.

Serum exosomes frequently are used for liquid biopsy. Serum exosomes normally are isolated using ultracentrifugation; however, ultracentrifugation is time-consuming, labor intensive and requires a high-speed centrifuge. Many commercial kits use a precipitation-based method; however, this process can result in substantial contamination. We developed a new method to isolate pure serum exosomes. We isolated serum exosomes using precipitation, extracted them using acetone, then isolated them again by precipitation. We used transmission electron microscopy (TEM) to examine the morphology of serum exosomes. TEM indicated that our isolated exosomes were pure with typical morphology and with a size ranging from 40 to 150 nm. Flow cytometry revealed expression of exosome markers, CD63, CA81 and CD9. Our double precipitation method enables ready extraction of pure exosomes from serum. Our double precipitation method simplifies detection of serum exosomal biomarkers for diagnosis and prognosis of disease.

Pro­differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts.

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p­Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho­rhino­phalangeal syndrome type I protein, zinc finger protein), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid 2­related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7­treated cells. Moreover, Fr7 counteracted H2O2­triggered cell damage, preventing increases in the pro­inflammatory and anti­chondrogenic microRNA (miR), miR­221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

Enhancing acetone production from H2 and CO2 using supplemental electron acceptors in an engineered Moorella thermoacetica.

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.

Micromycete Responses to Acetone: Electrochemical Express Evaluation of Acetone Metabolism.

The biosensor method has not yet been applied to study the fungus-acetone interaction. The first electrochemical (amperometric) study of Fusarium oxysporum f. sp. vasinfectum cells' responses to acetone was performed to evaluate the initial stages of the metabolism of acetone in the cells of the micromycete. Using a laboratory model of a membrane microbial sensor based on the micromycete cells, it was found that the fungus had constitutive enzyme systems that were involved in acetone transport into fungal cells. The research showed that uninduced by acetone cells had degradative activity against acetone. A positive cooperativity of acetone binding with enzymes initiating acetone degradation was revealed. Oxygen concentration affected the activation of cell enzymes initiating acetone degradation, but the cells' activity in the presence of acetone maintained even at low oxygen concentration. Kinetic parameters (the maximum rate of the cells' response to substrate and the half-saturation constant) of the processes causing the fungal cells' response to acetone were calculated. The results demonstrated the convenience of the biosensor method for assessing the potential of the micromycete as a substrate-degrading culture. In the future, the mechanism of response to acetone for microbial cells will be studied.

1'-O-methyl-averantin isolated from the endolichenic fungus Jackrogersella sp. EL001672 suppresses colorectal cancer stemness via sonic Hedgehog and Notch signaling.

Endolichenic fungi are host organisms that live on lichens and produce a wide variety of secondary metabolites. Colorectal cancer stem cells are capable of self-renewal and differentiation into cancer cells, which makes cancers difficult to eradicate. New alternative therapeutics are needed to inhibit the growth of tumor stem cells. This study examined the ability of an extract of Jackrogersella sp. EL001672 (derived from the lichen Cetraria sp.) and the isolated compound 1'-O-methyl-averantin to inhibit development of cancer stemness. The endolichenic fungus Jackrogersella sp. EL001672 (KACC 83021BP), derived from Cetraria sp., was grown in culture medium. The culture broth was extracted with acetone to obtain a crude extract. Column chromatography and reverse-phase HPLC were used to isolate an active compound. The anticancer activity of the extract and the isolated compound was evaluated by qRT-PCR and western blotting, and in cell viability, spheroid formation, and reporter assays. The acetone extract of EL001672 did not affect cell viability. However, 1'-O-methyl-averantin showed cytotoxic effects against cancer cell lines at 50 µg/mL and 25 µg/mL. Both the crude extract and 1'-O-methyl-averantin suppressed spheroid formation in CRC cell lines, and downregulated expression of stemness markers ALDH1, CD44, CD133, Lgr-5, Msi-1, and EphB1. To further characterize the mechanism underlying anti-stemness activity, we examined sonic Hedgehog and Notch signaling. The results showed that the crude extract and the 1'-O-methyl-averantin inhibited Gli1, Gli2, SMO, Bmi-1, Notch-1, Hes-1, and the CSL complex. Consequently, an acetone extract and 1'-O-methyl-averantin isolated from EL001672 suppresses colorectal cancer stemness by regulating the sonic Hedgehog and Notch signaling pathways.

Current progress on engineering microbial strains and consortia for production of cellulosic butanol through consolidated bioprocessing.

In the last decades, fermentative production of n-butanol has regained substantial interest mainly owing to its use as drop-in-fuel. The use of lignocellulose as an alternative to traditional acetone-butanol-ethanol fermentation feedstocks (starchy biomass and molasses) can significantly increase the economic competitiveness of biobutanol over production from non-renewable sources (petroleum). However, the low cost of lignocellulose is offset by its high recalcitrance to biodegradation which generally requires chemical-physical pre-treatment and multiple bioreactor-based processes. The development of consolidated processing (i.e., single-pot fermentation) can dramatically reduce lignocellulose fermentation costs and promote its industrial application. Here, strategies for developing microbial strains and consortia that feature both efficient (hemi)cellulose depolymerization and butanol production will be depicted, that is, rational metabolic engineering of native (hemi)cellulolytic or native butanol-producing or other suitable microorganisms; protoplast fusion of (hemi)cellulolytic and butanol-producing strains; and co-culture of (hemi)cellulolytic and butanol-producing microbes. Irrespective of the fermentation feedstock, biobutanol production is inherently limited by the severe toxicity of this solvent that challenges process economic viability. Hence, an overview of strategies for developing butanol hypertolerant strains will be provided.

MXene-based wireless facemask enabled wearable breath acetone detection for lipid metabolic monitoring.

Breath acetone (BrAC) detection presents a promising scheme for noninvasive monitoring of metabolic health due to its close correlation to diets and exercise-regulated lipolysis. Herein, we report a Ti3C2Tx MXene-based wireless facemask for on-body BrAC detection and real-time tracking of lipid metabolism, where Ti3C2Tx MXene serves as a versatile nanoplatform for not only acetone detection but also breath interference filtration. The incorporation of in situ grown TiO2 and short peptides with Ti3C2Tx MXene further improves the acetone sensitivity and selectivity, while TiO2-MXene interfaces facilitate light-assisted response calibration. To further realize wearable breath monitoring, a miniaturized flexible detection tag has been integrated with a commercially available facemask, which enables facile BrAC detection and wireless data transmission. Through the hierarchically designed filtration-detection-calibration-transmission system, we realize BrAC detection down to 0.31 ppm (part per million) in breath. On-body breath tests validate the facemask in dynamically monitoring of lipid metabolism, which could guide dieter, athletes, and fitness enthusiasts to arrange diets and exercise activities. The proposed wearable platform opens up new possibility toward the practice of breath analysis as well as daily lipid metabolic management.

Multiple Receptors Contribute to the Attractive Response of Caenorhabditis elegans to Pathogenic Bacteria.

Nematodes feed mainly on bacteria and sense volatile signals through their chemosensory system to distinguish food from pathogens. Although nematodes recognizing bacteria by volatile metabolites are ubiquitous, little is known of the associated molecular mechanism. Here, we show that the antinematode bacterium Paenibacillus polymyxa KM2501-1 exhibits an attractive effect on Caenorhabditis elegans via volatile metabolites, of which furfural acetone (FAc) acts as a broad-spectrum nematode attractant. We show that the attractive response toward FAc requires both the G-protein-coupled receptors STR-2 in AWC neurons and SRA-13 in AWA and AWC neurons. In the downstream olfactory signaling cascades, both the transient receptor potential vanilloid channel and the cyclic nucleotide-gated channel are necessary for FAc sensation. These results indicate that multiple receptors and subsequent signaling cascades contribute to the attractive response of C. elegans to FAc, and FAc is the first reported ligand of SRA-13. Our current work discovers that P. polymyxa KM2501-1 exhibits an attractive effect on nematodes by secreting volatile metabolites, especially FAc and 2-heptanone, broadening our understanding of the interactions between bacterial pathogens and nematodes. IMPORTANCE Nematodes feed on nontoxic bacteria as a food resource and avoid toxic bacteria; they distinguish them through their volatile metabolites. However, the mechanism of how nematodes recognize bacteria by volatile metabolites is not fully understood. Here, the antinematode bacterium Paenibacillus polymyxa KM2501-1 is found to exhibit an attractive effect on Caenorhabditis elegans via volatile metabolites, including FAc. We further reveal that the attractive response of C. elegans toward FAc requires multiple G-protein-coupled receptors and downstream olfactory signaling cascades in AWA and AWC neurons. This study highlights the important role of volatile metabolites in the interaction between nematodes and bacteria and confirms that multiple G-protein-coupled receptors on different olfactory neurons of C. elegans can jointly sense bacterial volatile signals.

Biobutanol production from sustainable biomass process of anaerobic ABE fermentation for industrial applications.

The growing population increases the need to develop advanced biological methods for utilizing renewable and sustainable resources to produce environmentally friendly biofuels. Currently, energy resources are limited for global demand and are constantly depleting and creating environmental problems. Some higher chain alcohols, like butanol and ethanol, processing similar properties to gasoline, can be alternate sources of biofuel. However, the industrial production of these alcohols remains challenging because they cannot be efficiently produced by microbes naturally. Therefore, butanol is the most interesting biofuel candidate with a higher octane number produced naturally by microbes through Acetone-Butanol-Ethanol fermentation. Feedstock selection as the substrate is the most crucial step in biobutanol production. Lignocellulosic biomass has been widely used to produce cellulosic biobutanol using agricultural wastes and residue. Specific necessary pretreatments, fermentation strategies, bioreactor designing and kinetics, and modeling can also enhance the efficient production of biobutanol. The recent genetic engineering approaches of gene knock in, knock out, and overexpression to manipulate pathways can increase the production of biobutanol in a user friendly host organism. So far various genetic manipulation techniques like antisense RNA, TargeTron Technology and CRISPR have been used to target Clostridium acetobutylicum for biobutanol production. This review summarizes the recent research and development for the efficient production of biobutanol in various aspects.

On the Mechanism of Selective Chemical Exchange of Bacteriopheophytins in the Reaction Centers of Rhodobacter sphaeroides R-26.

To elucidate the mechanism of site-selective chemical replacement of chromophores in the reaction centers (RCs) of photosynthetic bacteria by external pigments, we investigated how the efficiency of incorporation of plant pheophytin a (Pheo) into the binding sites for bacteriopheophytin a molecules (BPheo) in the isolated Rhodobacter sphaeroides R-26 RCs depended on the incubation medium temperature, Pheo aggregation state, and the presence of organic solvent (acetone). When Pheo was in a form of monomers in free detergent micelles in a water-detergent incubation medium, the degree of selective replacement of photochemically inactive BPheo HB molecules upon incubation of the RC/Pheo mixture at 5°C was ~15%. The exchange efficiency increased to 40% upon incubation at 25°C and reached 100% at the same temperature when 10% acetone was added to the incubation medium. At both 5 and 25°C, the degree of pigment exchange increased approximately twice, when a mixture of Pheo monomers and dimers in the presence of 10% acetone was used as the incubation medium. The removal of acetone from this medium with the preservation of pigment forms led to a significant decrease in the efficiency of Pheo incorporation. The effect of acetone on the pigment exchange was also observed at an elevated incubation temperature (43.5°C), when functionally active BPheo HA molecules were partially replaced. The results are discussed in terms of the mechanism according to which (i) the temperature-dependent internal movements of the RC protein facilitate the release of the BPheo molecule from the binding site with simultaneous insertion of the Pheo molecule into the same site in a coupled process, (ii) the role of temperature largely depends on the steric accessibility of binding pockets in the RC protein, (iii) the incorporation of Pheo occurs from a pool of monomeric molecules included in the RC-detergent micelles, and (iv) the presence of acetone in the incubation medium facilitates the exchange of Pheo monomers between micelles in the solution and the detergent belt of the RC complex.

Occupational exposure assessment with solid substances: choosing a vehicle for in vitro percutaneous absorption experiments.

Percutaneous occupational exposure to industrial toxicants can be assessed in vitro on excised human or animal skins. Numerous factors can significantly influence skin permeation of chemicals and the flux determination. Among them, the vehicle used to solubilize the solid substances is a tricky key step. A "realistic surrogate" that closely matches the exposure scenario is recommended in first intention. When direct transposition of occupational exposure conditions to in vitro experiments is impossible, it is recommended that the vehicle used does not affect the skin barrier (in particular in terms of structural integrity, composition, or enzymatic activity). Indeed, any such effect could alter the percutaneous absorption of substances in a number of ways, as we will see. Potential effects are described for five monophasic vehicles, including the three most frequently used: water, ethanol, acetone; and two that are more rarely used, but are realistic: artificial sebum and artificial sweat. Finally, we discuss a number of criteria to be verified and the associated tests that should be performed when choosing the most appropriate vehicle, keeping in mind that, in the context of occupational exposure, the scientific quality of the percutaneous absorption data provided, and how they are interpreted, may have long-range consequences. From the narrative review presented, we also identify and discuss important factors to consider in future updates of the OECD guidelines for in vitro skin absorption experiments.

Co-expression of an isopropanol synthetic operon and eGFP to monitor the robustness of Cupriavidus necator during isopropanol production.

Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol/acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighted the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered.

Proanthocyanidins Modulate Rumen Enzyme Activities and Protein Utilization In Vitro.

This study investigated the principal leaf protein (rubisco) solubilization and in vitro ruminal enzyme activity in relation to the molecular structure of proanthocyanidins extracted from leaves of Anogeissus pendula and Eugenia jambolana. Six proanthocyanidin fractions were extracted by 50% (v/v) methanol−water followed by 70% (v/v) acetone−water and then distilled water from leaves of A. pendula (AP) and E. jambolana (EJ) to yield EJ−70, EJ−50, EJ−DW, AP−70, AP−50 and AP−DW. Fractions were examined for their molecular structure and their effects on sheep ruminal enzymes and solubilization of rubisco in vitro. All fractions significantly (p < 0.05) inhibited the activity of ruminal glutamic oxaloacetic transaminase and glutamic pyruvic transaminase. The fractions AP−50 and EJ−50 significantly inhibited the activity of the R-cellulase enzyme. Most of the fractions inhibited R-glutamate dehydrogenase activity (p < 0.05) by increasing its concentration, while protease activity decreased by up to 58% with increasing incubation time and concentration. The solubilization of rubisco was observed to be comparatively higher in A. pendula (16.60 ± 1.97%) and E. jambolana (15.03 ± 1.06%) than that of wheat straw (8.95 ± 0.95%) and berseem hay (3.04 ± 0.08%). A significant (p < 0.05) increase in protein solubilization was observed when wheat straw and berseem hay were supplemented with A. pendula and E. jambolana leaves at different proportions. The efficiency of microbial protein was significantly (p < 0.05) greater with the supplementation of leaves of A. pendula in comparison to E. jambolana. The overall conclusion is that the proanthocyanidins obtained from E. jambolana exhibited greater inhibitory activities on rumen enzymes, whereas A. pendula recorded higher protein solubilization. Thus, PAs from A. pendula and E. jambolana appear to have the potential to manipulate rumen enzyme activities for efficient utilization of protein and fiber in ruminants.