Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica.

BACKGROUND: Oleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches. RESULTS: To demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids were susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved. The production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons shown in previous studies could be confirmed. CONCLUSIONS: Oleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 mL bioreactors. The lighting turned out to be a crucial factor in terms of growth and hydrocarbon production, therefore, the evaluation of different conditions was an important step towards a tailor-made process. In general, the developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from being substrates for further enzymatic or chemical modification or as a drop-in biofuel blend.

A comparison of stability between delayed versus immediately loaded orthodontic palatal implants.

INTRODUCTION: Control of anchorage is a fundamental problem in orthodontics. Conventional means of controlling anchorage are characterized by potential disadvantages and inconveniences: visibility, compliance dependence, risk of undesirable side effects, and injury. Titanium implants have evolved as a potential clinical alternative in overcoming the limits of conventional dental orthodontic anchorage. METHODS: This project was designed as a prospective observational study on 20 patients whose treatment plans required maximum (stable) anchorage during orthodontic treatment. The patients received palatal implants (Institut Straumann AG, Waldenburg, Switzerland: length of implant 4-6 mm, diameter 3.3 mm), which were placed into the midpalate. The goal of this study was to evaluate if the implant could be loaded immediately, or if time should be allowed for integration. Patients were randomized into two groups; one group had their implants loaded immediately with a coil spring, and the second group remained nonloaded, with an annealed coil spring, for the 8-week experimental period. Measurement of implant stability was taken using resonance frequency analysis on both groups at the time of implant placement and at 8 weeks post-placement. RESULTS: This study demonstrated that immediate loading of the Straumann orthodontic implant is possible, based on the clinical success observed in both groups. However, compared with the nonloaded group, the stability of the immediately loaded implant was significantly less at 8 weeks. The mean implant stability quotient (ISQ) of the nonloaded group was 38.7 kHz at baseline and 47.3 kHz after 8 weeks. The mean ISQ of the loaded group was 42.0 kHz at baseline and 38.4 kHz after 8 weeks. Statistical analysis showed a significant difference between the group that was loaded and the nonloaded group after 8 weeks (p < 0.05). CONCLUSION: Based on the results of this study, an unloaded healing period provides for increased stability of the implants compared with immediately loaded palatal implants. CLINICAL SIGNIFICANCE: Patients often like to have their orthodontic treatment begin as soon as possible. This study examined if palatal implants could be loaded immediately after placement so overall treatment time could be decreased. It appears that this is possible based on the results of the study; however, an unloaded healing period results in a more stable implant.

MMP-2 and MMP-9 and their tissue inhibitors in the plasma of preterm and term neonates.

Matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) are involved in a variety of physiologic growth and development and pathophysiologic inflammatory conditions. We hypothesized that 1) MMP-2 and -9 plasma activities and TIMP-1 and -2 plasma concentrations in preterm and term neonates were dependent on the gestational and postnatal age; and 2) the respective MMP and their inhibitors were deranged in the development of bronchopulmonary dysplasia (BPD) and intraventricular hemorrhage (IVH) in preterm neonates. From 1998 to 1999, blood samples were collected from preterm neonates (25-36 wk gestation) with or without BPD and/or IVH as well as from healthy term (37-40 wk gestation) neonates during the first 28 d of life. MMP-2 and MMP-9 plasma activities were measured by zymography; TIMP-1 and TIMP-2 plasma concentrations were determined by ELISA. In neonates without BPD or IVH (n = 50), MMP-2 and MMP-9 plasma activities both appeared to be gestational age dependent, with the highest levels observed in neonates of 33-36 wk gestation. TIMP-1 plasma concentration was highest in term neonates but no gestational difference was found in TIMP-2. Only MMP-9 showed a 50% decrease after d 1 in the first postnatal month. Twelve preterm infants with BPD and/or IVH had significantly lower MMP-2 but higher MMP-9 activity and higher TIMP-1 concentration than those of corresponding neonates without BPD or IVH. These findings show the gestational age-dependent expression of plasma MMP activities and their inhibitors. MMP and TIMP may be involved in the feto-neonatal development and may contribute to the pathogenesis of BPD and/or IVH in critically ill preterm neonates.

Expression and activity of matrix metallo proteinases 2 and 9 and their inhibitors in rat lungs during the perinatal period and in diaphragmatic hernia.

During lung development, the extracellular matrix undergoes dynamic remodeling. Matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinases (TIMPs), are important enzymes that participate in regulating tissue remodeling. There is an abnormal balance of the synthesis and degradation of collagen and elastin in perinatal lung associated with congenital diaphragmatic hernia (CDH). This study was designed to (1) determine the expression and gelatinolytic activity patterns of MMPs 2 and 9 and TIMPs 1 and 2 in rat lungs during the perinatal period, and (2) to test the hypothesis that they are abnormal in nitrofen-induced CDH. Measurements were made using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and zymography. The mRNA expression and activity of MMP 2 did not change significantly from embryonic day 16 to postnatal day 14. The most striking feature found was the rapid increase in the expression of MMP 9 soon after birth. Measurements were repeated on lung tissue isolated from embryonic rats with nitrofen-induced CDH. The expression and activity of MMPs and TIMPs were similar to control values and thus we conclude that these proteins appear not to be responsible for the altered extracellular matrix and morphological abnormalities noted in CDH lungs at birth.