Dynamic flux balance analysis of 1,3-propanediol production by clostridium butyricum fermentation.

To study the relationship between the yield of 1,3-propanediol (1,3-PDO) and the flux change of the Clostridium butyricum metabolic pathway, an optimized calculation method based on dynamic flux balance analysis was used by combining genome-scale flux balance analysis with a kinetic model. A more comprehensive and extensive metabolic pathway was obtained by optimization calculations. The primary extended branches include: the dihydroxyacetone node, which enters the pentose phosphate pathway; the α-oxoglutarate node, which has synthetic metabolic pathways for glutamic acid and amino acids; and the serine and homocysteine nodes, which produce cystathionine before homocysteine enters the methionine cycle pathway. According to the expanded metabolic network, the flux distribution of key nodes in the metabolic pathway and the relationship between the flux distribution ratio of nodes and the yield of 1,3-PDO were analyzed. At the dihydroxyacetone node, the flux of dihydroxyacetone converted to dihydroxyacetone phosphate was positively correlated with the yield of 1,3-PDO. As an important intermediate product, the flux change in the metabolic pathway of α-oxoglutarate reacting with amino acids to produce glutamic acid is positively correlated with the yield. When pyruvate was used as the central node to convert into lactic acid and α-oxoglutarate, the proportion of branch flux was negatively correlated with the yield of 1,3-PDO. These studies provide a theoretical basis for the optimization and further study of the metabolic pathway of C. butyricum.

Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield.

Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.

Acute and subacute oral toxicity of propylene glycol enantiomers in mice and the underlying nephrotoxic mechanism.

Propylene glycol (PG; 1,2-propanediol) has been commonly used as a food additive and vehicle in pharmaceutical preparations. PG can form rectus (R-) enantiomers and sinister (S-) enantiomers. Herein, Kunming mice were used as the animal model to evaluate the acute and subacute oral toxicity of R-PG, S-PG and RS-PG (1:1 racemic mixture of R-PG and S-PG). The median lethal doses of R-PG, S-PG and RS-PG administered by oral gavage to mice were 22.81 g/kg, 26.62 g/kg and 24.92 g/kg, respectively. In the 28-day oral subacute toxicity study, the body weight, organ weights, serum biochemical, and renal histology were examined. There was no difference in subacute toxicity among R-PG, S-PG and RS-PG. The administration of 1 and 5 g/kg/day PG for 28 days caused nephrotoxicity. The kidney somatic index and levels of blood urea nitrogen exhibited a significant increase. Moreover, the activities of superoxide dismutase, catalase, and glutathione peroxidase significantly decreased after the treatment with PG. The levels of malondialdehyde, tumor necrosis factor α, interleukin 1ß, and interleukin 6 significantly increased in the kidney. The results show that the nephrotoxic effects of PG are induced by oxidative stress, and the activation of the inflammatory response is mediated by the NF-κB signaling pathway. Together, these findings provide information on R-PG, S-PG and RS-PG treatments for the risk assessment of toxicity and effects on human health.

Genetic Characterization of a Glycyl Radical Microcompartment Used for 1,2-Propanediol Fermentation by Uropathogenic Escherichia coli CFT073.

Bacterial microcompartments (MCPs) are widespread protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of MCPs is to optimize metabolic pathways by confining toxic and/or volatile pathway intermediates. A major class of MCPs known as glycyl radical MCPs has only been partially characterized. Here, we show that uropathogenic Escherichia coli CFT073 uses a glycyl radical MCP for 1,2-propanediol (1,2-PD) fermentation. Bioinformatic analyses identified a large gene cluster (named grp for glycyl radical propanediol) that encodes homologs of a glycyl radical diol dehydratase, other 1,2-PD catabolic enzymes, and MCP shell proteins. Growth studies showed that E. coli CFT073 grows on 1,2-PD under anaerobic conditions but not under aerobic conditions. All 19 grp genes were individually deleted, and 8/19 were required for 1,2-PD fermentation. Electron microscopy and genetic studies showed that a bacterial MCP is involved. Bioinformatics combined with genetic analyses support a proposed pathway of 1,2-PD degradation and suggest that enzymatic cofactors are recycled internally within the Grp MCP. A two-component system (grpP and grpQ) is shown to mediate induction of the grp locus by 1,2-PD. Tests of the E. coli Reference (ECOR) collection indicate that >10% of E. coli strains ferment 1,2-PD using a glycyl radical MCP. In contrast to other MCP systems, individual deletions of MCP shell genes (grpE, grpH, and grpI) eliminated 1,2-PD catabolism, suggesting significant functional differences with known MCPs. Overall, the studies presented here are the first comprehensive genetic analysis of a Grp-type MCP.IMPORTANCE Bacterial MCPs have a number of potential biotechnology applications and have been linked to bacterial pathogenesis, cancer, and heart disease. Glycyl radical MCPs are a large but understudied class of bacterial MCPs. Here, we show that uropathogenic E. coli CFT073 uses a glycyl radical MCP for 1,2-PD fermentation, and we conduct a comprehensive genetic analysis of the genes involved. Studies suggest significant functional differences between the glycyl radical MCP of E. coli CFT073 and better-studied MCPs. They also provide a foundation for building a deeper general understanding of glycyl radical MCPs in an organism where sophisticated genetic methods are available.

Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage.

Vitrification is a cryopreservation technique for the long-term storage of viable tissue, but the success of this technique relies on multiple factors. In 2012, our group published a working vitrification protocol for intact human articular cartilage and reported promising chondrocyte recovery after using a four-step multi-cryoprotectant (CPA) loading method that required 570 min. However, this protocol requires further optimization for clinical practice. Herein, we compared three multi-step CPA loading protocols to investigate their impact on chondrocyte recovery after vitrification of porcine articular cartilage on a bone base, including our previous four-step protocol (original: 570 min), and two shorter three-step protocols (optimized: 420 min, and minimally vitrifiable: 310 min). Four different CPAs were used including glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. As vitrification containers, two conical tubes (50 ml and 15 ml) were evaluated for their heat transfer impact on chondrocyte recovery after vitrification. Osteochondral dowels were cored into two diameters of 10.0 mm and 6.9 mm with an approximately 10-mm thick bone base, and then allocated into the twelve experimental groups based on CPA loading protocol, osteochondral dowel size, and vitrification container size. After vitrification at -196 °C and tissue warming and CPA removal, samples in all groups were assessed for both chondrocyte viability and metabolic activity. The optimized protocol proposed based on mathematical modelling resulted in similar chondrocyte recovery to our original protocol and it was 150 min shorter. Furthermore, this study illustrated the role of CPA permeation (dowel size) and heat transfer (container size) on vitrification protocol outcome.

Variations in coil temperature/power and e-liquid constituents change size and lung deposition of particles emitted by an electronic cigarette.

Electronic cigarette uses propylene glycol and glycerol to deliver nicotine and flavors to the lungs. Given the hundreds of different brands, the thousands of flavors available and the variations in nicotine concentrations, it is likely that electronic cigarette settings and e-liquid composition affect the size distribution of particles emitted and ultimately pulmonary deposition. We used the inExpose e-cigarette extension to study two separate modes of operation of electronic cigarettes, namely power-controlled and the temperature-controlled. We also assessed several e-liquids based on propylene glycol and glycerol concentrations, nicotine content, and selected monomolecular flavoring agents (menthol, vanillin, and maltol). Particle size distribution was measured using a Condensation Particle Counter and a Scanning Mobility Particle Sizer spectrometer. Lung deposition was predicted using the International Commission on Radiological Protection model. For all resistance coils, increase in power delivery generated larger particles while maintaining a higher coil temperature generated smaller particles. Increase in glycerol concentration led to the generation of larger particles. With regard to flavors, we showed that despite minor effect of menthol and maltol, vanillin dramatically increased particle size. Presence of nicotine also increased particle size. Finally, particles emitted by the electronic cigarette were predicted to mainly deposit in the alveoli and conditions generating larger particle sizes led to a reduction in predicted lung deposition. This study shows that coil temperature, propylene glycol and glycerol concentrations, presence of nicotine, and flavors affect the size of particles emitted by an electronic cigarette, directly affecting predicted lung deposition of these particles.

Metabolic adaptation of adherent-invasive Escherichia coli to exposure to bile salts.

The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of Crohn's disease patients, adhere to intestinal epithelial cells, invade them and exacerbate intestinal inflammation. The high nutrient competition between the commensal microbiota and AIEC pathobiont requires the latter to occupy their own metabolic niches to survive and proliferate within the gut. In this study, a global RNA sequencing of AIEC strain LF82 has been used to observe the impact of bile salts on the expression of metabolic genes. The results showed a global up-regulation of genes involved in degradation and a down-regulation of those implicated in biosynthesis. The main up-regulated degradation pathways were ethanolamine, 1,2-propanediol and citrate utilization, as well as the methyl-citrate pathway. Our study reveals that ethanolamine utilization bestows a competitive advantage of AIEC strains that are metabolically capable of its degradation in the presence of bile salts. We observed that bile salts activated secondary metabolism pathways that communicate to provide an energy benefit to AIEC. Bile salts may be used by AIEC as an environmental signal to promote their colonization.

A toxicity cost function approach to optimal CPA equilibration in tissues.

There is growing need for cryopreserved tissue samples that can be used in transplantation and regenerative medicine. While a number of specific tissue types have been successfully cryopreserved, this success is not general, and there is not a uniform approach to cryopreservation of arbitrary tissues. Additionally, while there are a number of long-established approaches towards optimizing cryoprotocols in single cell suspensions, and even plated cell monolayers, computational approaches in tissue cryopreservation have classically been limited to explanatory models. Here we develop a numerical approach to adapt cell-based CPA equilibration damage models for use in a classical tissue mass transport model. To implement this with real-world parameters, we measured CPA diffusivity in three human-sourced tissue types, skin, fibroid and myometrium, yielding propylene glycol diffusivities of 0.6 × 10-6 cm2/s, 1.2 × 10-6 cm2/s and 1.3 × 10-6 cm2/s, respectively. Based on these results, we numerically predict and compare optimal multistep equilibration protocols that minimize the cell-based cumulative toxicity cost function and the damage due to excessive osmotic gradients at the tissue boundary. Our numerical results show that there are fundamental differences between protocols designed to minimize total CPA exposure time in tissues and protocols designed to minimize accumulated CPA toxicity, and that "one size fits all" stepwise approaches are predicted to be more toxic and take considerably longer than needed.

Enantioselective uptake, translocation and degradation of the chiral pesticides tebuconazole and imazalil by Phragmites australis.

Phytoremediation of realistic environmental concentrations (10 µg L-1) of the chiral pesticides tebuconazole and imazalil by Phragmites australis was investigated. This study focussed on removal dynamics, enantioselective mechanisms and transformation products (TPs) in both hydroponic growth solutions and plant tissues. For the first time, we documented uptake, translocation and metabolisation of these pesticides inside wetland plants, using enantioselective analysis. Tebuconazole and imazalil removal efficiencies from water reached 96.1% and 99.8%, respectively, by the end of the experiment (day 24). Removal from the solutions could be described by first-order removal kinetics with removal rate constants of 0.14 d-1 for tebuconazole and 0.31 d-1 for imazalil. Removal of the pesticides from the hydroponic solution, plant uptake, within plant translocation and degradation occurred simultaneously. Tebuconazole and imazalil concentrations inside Phragmites peaked at day 10 and 5d, respectively, and decreased thereafter. TPs of tebuconazole i.e., (5-(4-Chlorophenyl)-2,2-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)-1,3-pentanediol and 5-(3-((1H-1,2,4-Triazol-1-yl)methyl)-3-hydroxy-4,4-dimethylpentyl)-2-chlorophenol) were quantified in solution, while the imazalil TPs (α-(2,4-Dichlorophenyl)-1H-imidazole-1-ethanol and 3-[1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethoxy]-1,2-propanediol) were quantified in both solution and plant tissue. Pesticide uptake by Phragmites was positively correlated with evapotranspiration. Pesticide removal from the hydroponic solution was not enantioselective. However, tebuconazole was degraded enantioselectively both in the roots and shoots. Imazalil translocation and degradation inside Phragmites were also enantioselective: R-imazalil translocated faster than S-imazalil.

Evaluation of the ability to metabolize 1, 2-propanediol by heterofermentative bacteria of the genus Lactobacillus

Background: New directions of research on lactic acid bacteria include investigation of metabolic pathways for the synthesis and/or metabolism of 1,2-propanediol, commonly used in the food and chemical industry, medicine, pharmacy and cosmetology as well as agriculture. The objective of this study was to compare the capacity of strains representing three diverse heterofermentative species belonging to the genus Lactobacillus to synthesize and/or transform 1,2-PD as well as to suggest new directions of research aimed at commercial use of this metabolite. Results: The novel strain of Lactobacillus buchneri A KKP 2047p, characterized as exhibiting an unusual trait for that species in the form of capacity to metabolize 1,2-PD, grew poorly in a medium containing 1,2-PD as a sole carbon source. The supplementation with glucose facilitated rapid growth of bacteria and use of 1,2-PD for the synthesis of propionic acid. A similar observation was noted for Lactobacillus reuteri. On the other hand, Lactobacillus diolivorans effectively metabolized 1,2-PD which was the sole carbon source in the medium, and the addition of glucose inhibited the synthesis of propionic acid. The experiments also investigated the effect of cobalamin as a diol dehydratase coenzyme involved in the propionic acid synthesis from 1,2-PD whose addition promoted the yield of the reaction in the case of all tested strains. Conclusions: All tested isolates showed the ability to effectively metabolize 1,2-PD (in the presence of cobalamin) and its conversion to propionic acid, which reveals that investigated bacteria meet the essential requirements of microorganisms with a potential application.

In vitro human epidermal permeation of nicotine from electronic cigarette refill liquids and implications for dermal exposure assessment.

Nicotine plus flavorings in a propylene glycol (PG) vehicle are the components of electronic cigarette liquids (e-liquids), which are vaporized and inhaled by the user. Dermal exposure to nicotine and e-liquids may occur among workers in mixing and filling of e-cigarettes in the manufacturing process. Inadvertent skin contact among consumers is also a concern. In vitro nicotine permeation studies using heat-separated human epidermis were performed with surrogate and two commercial e-liquids, neat and aqueous nicotine donor formulations. Steady-state fluxes (Jss), and lag times (tlag) were measured for each formulation. In addition, transient (4 h) exposure and finite dose (1-10 µl/cm2) experiments were undertaken using one commercial e-liquid. Average Jss (µg/cm2/h) from formulations were: nicotine in PG (24 mg/ml): 3.97; commercial e-liquid containing menthol (25 mg/ml nicotine): 10.2; commercial e-liquid containing limonene (25 mg/ml nicotine): 23.7; neat nicotine: 175. E-liquid lag times ranged from 5 to 10 h. Absorbed fraction of nicotine from finite doses was ≈0.3 at 48 h. The data were applied to transient exposure and finite dose dermal exposure assessment models and to a simple pharmacokinetic model. Three illustrative exposure scenarios demonstrate use of the data to predict systemic uptake and plasma concentrations from dermal exposure. The data demonstrate the potential for significant nicotine absorption through skin contact with e-cigarette refill solutions and the neat nicotine used to mix them.

Perfil eletroforético do colostro de ovelhas suplementadas com propileno glicol e cobalto associado à vitamina B12 no final da gestação / Electrophoretic profile of the colostrum of ewes supplemented with propylene glycol and cobalt associated with vitamin B12 in late pregnancy

Este trabalho teve por objetivo avaliar o proteinograma do colostro de ovelhas submetidas a administração de propileno glicol e de cobalto associado à vitamina B12 no final da gestação. Dezoito ovelhas da raça Santa Inês, prenhas e com idade variando entre 18 meses a cinco anos foram distribuídas, por amostragem probabilística em três grupos experimentais, aproximadamente 30 dias antes da data prevista para o parto. No Grupo 1 (G1/n=6) foram administrados 30mL de propileno glicol P.A. via oral diariamente; no Grupo 2 (G2/n=6) foi administrado 1mg de cloreto de cobalto em solução a 1% via oral diariamente e 2mg de vitamina B12, via intramuscular semanalmente e no Grupo 3 (G3/n=6): grupo controle. Logo após o parto procedeu-se a colheita de 30mL de colostro, que foram acondicionados em recipientes apropriados e encaminhados ao laboratório. Após homogeneização, adicionou-se a cada 1.000µL de colostro, 75µL de solução de renina, que foi mantido em banho-maria a 37ºC por aproximadamente 20 minutos e centrifugado a 21.000G durante dez minutos em centrífuga refrigerada. Posteriormente, a fração intermediária, correspondente ao soro do colostro, foi aliquotada e mantida em ultrafreezer a -80oC para posterior determinação das proteínas. A determinação da proteína total do soro colostral foi realizada empregando-se reagente comercial. A separação das proteínas foi realizada utilizando-se a técnica de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE). Foram identificadas as proteínas IgA, lactoferrina, albumina, IgG de cadeia pesada (IgGCP), ß-caseína, IgG de cadeia leve (IgGCL), ß-lactoglobulina and α-lactoalbumina, não havendo influência da administração dos suplementos na fase final da gestação sobre as concentrações protéicas do colostro.(AU)

This study aimed to evaluate the proteinogram of the colostrum of ewes submitted to administration of propylene glycol and cobalt associated with vitamin B12 in late pregnancy. Eighteen pregnant Santa Inês ewes 18 months to 5 years old were distributed by probabilistic sampling into three experimental groups, about 30 days before the expected delivery date. In group 1 (G1/n=6), daily oral doses of 30ml propylene glycol PA were administered; Group 2 (G2/n=2) received a daily oral dosage of 1mg cobalt chloride in 1% solution and 2mg of vitamin B12 intramuscularly once a week, and Group 3 (G3/n=6) was the control group. Soon after delivery 30mL of colostrum was harvested from each ewe, which were stored in appropriate containers and sent to the laboratory. After homogenization, we added to each 1000µL of colostrum 75µL solution of rennin, which was kept in a water bath at 37°C for about 20 minutes and centrifuged at 21.000G for 10 minutes in a refrigerated centrifuge. Later, the intermediate fraction, corresponding to colostrum whey, was aliquoted and kept in a -80oC ultrafreezer for subsequent determination of proteins. The determination of the total colostral protein whey was performed using a commercial reagent, observing the linearity test for colostrum. The separation of proteins was performed using the technique of electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). Lactoferrin, IgA, albumin, IgG heavy chain (IgGCP), ß-casein, IgG light chain (IgGCL), ß-lactoglobulin and α-lactalbumin proteins were identified. There was no influence of the administration of supplements during late pregnancy on the concentration of proteins identified in the colostrum of the ewes.(AU)

An enantioselective NADP(+)-dependent alcohol dehydrogenase responsible for cooxidative production of (3S)-5-hydroxy-3-methyl-pentanoic acid.

A soil bacterium, Mycobacterium sp. B-009, is able to grow on racemic 1,2-propanediol (PD). The strain was revealed to oxidize 3-methyl-1,5-pentanediol (MPD) to 5-hydroxy-3-methyl-pentanoic acid (HMPA) during growth on PD. MPD was converted into an almost equimolar amount of the S-form of HMPA (S-HMPA) at 72%ee, suggesting the presence of an enantioselective MPD dehydrogenase (MPD-DH). As expected, an NADP(+)-dependent alcohol dehydrogenase, which catalyzes the initial step of MPD oxidation, was detected and purified from the cell-free extract. This enzyme was suggested to be a homodimeric medium-chain alcohol dehydrogenase/reductase (MDR). The catalytic and kinetic parameters indicated that MPD is the most suitable substrate for the enzyme. The enzyme was encoded by a 1047-bp gene (mpd1) and several mycobacterial strains were found to have putative MDR genes similar to mpd1. In a phylogenetic tree, MPD-DH formed an independent clade together with the putative MDR of Mycobacterium neoaurum, which produces opportunistic infections.

A study of the osmotic characteristics, water permeability, and cryoprotectant permeability of human vaginal immune cells.

Cryopreservation of specimens taken from the genital tract of women is important for studying mucosal immunity during HIV prevention trials. However, it is unclear whether the current, empirically developed cryopreservation procedures for peripheral blood cells are also ideal for genital specimens. The optimal cryopreservation protocol depends on the cryobiological features of the cells. Thus, we obtained tissue specimens from vaginal repair surgeries, isolated and flow cytometry-purified immune cells, and determined fundamental cryobiological characteristics of vaginal CD3(+) T cells and CD14(+) macrophages using a microfluidic device. The osmotically inactive volumes of the two cell types (Vb) were determined relative to the initial cell volume (V0) by exposing the cells to hypotonic and hypertonic saline solutions, evaluating the equilibrium volume, and applying the Boyle van't Hoff relationship. The cell membrane permeability to water (Lp) and to four different cryoprotective agent (CPA) solutions (Ps) at room temperature were also measured. Results indicated Vb values of 0.516 V0 and 0.457 V0 for mucosal T cells and macrophages, respectively. Lp values at room temperature were 0.196 and 0.295 µm/min/atm for T cells and macrophages, respectively. Both cell types had high Ps values for the three CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) (minimum of 0.418 × 10(-3) cm/min), but transport of the fourth CPA, glycerol, occurred 50-150 times more slowly. Thus, DMSO, PG, and EG are better options than glycerol in avoiding severe cell volume excursion and osmotic injury during CPA addition and removal for cryopreservation of human vaginal immune cells.

Nicotine delivery, retention and pharmacokinetics from various electronic cigarettes.

AIMS: To measure the systemic retention of nicotine, propylene glycol (PG) and vegetable glycerin (VG) in electronic cigarette (e-cigarette) users, and assess the abuse liability of e-cigarettes by characterizing nicotine pharmacokinetics. DESIGN: E-cigarette users recruited over the internet participated in a 1-day research ward study. Subjects took 15 puffs from their usual brand of e-cigarette. Exhaled breath was trapped in gas-washing bottles and blood was sampled before and several times after use. SETTING: San Francisco, California, USA. PARTICIPANTS: Thirteen healthy, experienced adult e-cigarette users (six females and seven males). MEASUREMENTS: Plasma nicotine was analyzed by gas chromatography-mass spectrometry (GC-MS/MS) and nicotine, VG and PG in e-liquids and gas traps were analyzed by LC-MS/MS. Heart rate changes and subjective effects were assessed. FINDINGS: E-cigarettes delivered an average of 1.33 (0.87-1.79) mg [mean and 95% confidence interval (CI)] of nicotine, and 93.8% of the inhaled dose, 1.22 (0.80-1.66) was systemically retained. Average maximum plasma nicotine concentration (Cmax ) was 8.4 (5.4-11.5) ng/ml and time of maximal concentration (Tmax ) was 2-5 minutes. One participant had Tmax of 30 minutes. 84.4% and 91.7% of VG and PG, respectively, was systemically retained. Heart rate increased by an average of 8.0 beats per minute after 5 minutes. Withdrawal and urge to smoke decreased and the e-cigarettes were described as satisfying. CONCLUSIONS: E-cigarettes can deliver levels of nicotine that are comparable to or higher than typical tobacco cigarettes, with similar systemic retention. Although the average maximum plasma nicotine concentration in experienced e-cigarette users appears to be generally lower than what has been reported from tobacco cigarette use, the shape of the pharmacokinetic curve is similar, suggesting addictive potential.

Systematically engineering Escherichia coli for enhanced production of 1,2-propanediol and 1-propanol.

The biological production of high value commodity 1,2-propanediol has been established by engineering the glycolysis pathway. However, the simultaneous achievement of high titer and high yield has not been reported yet, as all efforts in increasing the titer have resulted in low yields. In this work, we overcome this limitation by employing an optimal minimal set of enzymes, channeling the carbon flux into the 1,2-propanediol pathway, increasing NADH availability, and improving the anaerobic growth of the engineered Escherichia coli strain by developing a cell adaptation method. These efforts lead to 1,2-propanediol production at a titer of 5.13 g/L with a yield of 0.48 g/g glucose in 20 mL shake flask studies. On this basis, we pursue the enhancement of 1-propanol production from the 1,2-propanediol platform. By constructing a fusion diol dehydratase and developing a dual strain process, we achieve a 1-propanol titer of 2.91 g/L in 20 mL shake flask studies. To summarize, we report the production of 1,2-propanediol at enhanced titer and enhanced yield simultaneously in E. coli for the first time. Furthermore, we establish an efficient system for the production of biofuel 1-propanol biologically.

Fermentative production of 1-propanol from sugars using wild-type and recombinant Shimwellia blattae.

Shimwellia blattae is an enteric bacterium and produces endogenous enzymes that convert 1,2-propanediol (1,2-PD) to 1-propanol, which is expected to be used as a fuel substitute and a precursor of polypropylene. Therefore, if S. blattae could be induced to generate its own 1,2-PD from sugars, it might be possible to produce 1-propanol from sugars with this microorganism. Here, two 1,2-PD production pathways were constructed in S. blattae, resulting in two methods for 1-propanol production with the bacterium. One method employed the L-rhamnose utilization pathway, in which L-rhamnose is split into dihydroxyacetone phosphate and 1,2-PD. When wild-type S. blattae was cultured with L-rhamnose, an accumulation of 1,2-PD was observed. The other method for producing 1,2-PD was to introduce an engineered 1,2-PD production pathway from glucose into S. blattae. In both cases, the produced 1,2-PD was then converted to 1-propanol by 1,2-PD converting enzymes, whose production was induced by the addition of glycerol.

The N-terminal region of the medium subunit (PduD) packages adenosylcobalamin-dependent diol dehydratase (PduCDE) into the Pdu microcompartment.

Salmonella enterica produces a proteinaceous microcompartment for B(12)-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B(12)-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell.

Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella.

Hundreds of bacterial species use microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of a protein shell encapsulating specific metabolic enzymes. In Salmonella, an MCP is used for 1,2-propanediol utilization (Pdu MCP). The shell of this MCP is composed of eight different types of polypeptides, but their specific functions are uncertain. Here, we individually deleted the eight genes encoding the shell proteins of the Pdu MCP. The effects of each mutation on 1,2-PD degradation and MCP structure were determined by electron microscopy and growth studies. Deletion of the pduBB', pduJ, or pduN gene severely impaired MCP formation, and the observed defects were consistent with roles as facet, edge, or vertex protein, respectively. Metabolite measurements showed that pduA, pduBB', pduJ, or pduN deletion mutants accumulated propionaldehyde to toxic levels during 1,2-PD catabolism, indicating that the integrity of the shell was disrupted. Deletion of the pduK, pduT, or pduU gene did not substantially affect MCP structure or propionaldehyde accumulation, suggesting they are nonessential to MCP formation. However, the pduU or pduT deletion mutants grew more slowly than the wild type on 1,2-PD at saturating B(12), indicating that they are needed for maximal activity of the 1,2-PD degradative enzymes encased within the MCP shell. Considering recent crystallography studies, this suggests that PduT and PduU may mediate the transport of enzyme substrates/cofactors across the MCP shell. Interestingly, a pduK deletion caused MCP aggregation, suggesting a role in the spatial organization of MCP within the cytoplasm or perhaps in segregation at cell division.

Cryosolvent interaction with cellular actin using 3T3-L1cells as a model system.

Previous immunolocalisation studies using intact cells have identified modification of the cytoskeleton by cryoprotectants. In the present study we have used a proteomics approach to directly resolve the interactive effects of 3T3-L1cells exposed to two cryoprotectants, dimethyl sulphoxide (Me(2)SO) and 1,2-propanediol (PROH) in 5,10, 20 and 50(v/v) percent solutions, respectively. Two-dimensional protein electrophoresis and Western blot analysis of the cell extracts identified a range of immunoreactive actin fragments with varying molecular weights and isoelectric points at all cryoprotectant concentrations. The addition of either 10mM l-cysteine or reduced glutathione to the cells prior to cryprotectant exposure modified the actin fragmentation. In this preliminary report, we have provided direct evidence of actin fragmentation when exposed to cryoprotectants and have demonstrated that the use of redox agents can modify the cryprotectant action.