Isobutanol production from cellobiose in Escherichia coli

Converting lignocellulosics into biofuels remains a promising route for biofuel production. To facilitate strain development for specificity and productivity of cellulosic biofuel production, a user friendly Escherichia coli host was engineered to produce isobutanol, a drop-in biofuel candidate, from cellobiose. A beta-glucosidase was expressed extracellularly by either excretion into the media, or anchoring to the cell membrane. The excretion system allowed for E. coli to grow with cellobiose as a sole carbon source at rates comparable to those with glucose. The system was then combined with isobutanol production genes in three different configurations to determine whether gene arrangement affected isobutanol production. The most productive strain converted cellobiose to isobutanol in titers of 7.64?±?0.19 g/L with a productivity of 0.16 g/L/h. These results demonstrate that efficient cellobiose degradation and isobutanol production can be achieved by a single organism, and provide insight for optimization of strains for future use in a consolidated bioprocessing system for renewable production of isobutanol.     

Bacteriophage Ø105clz induces the GroEL-homologue protein inBacillus subtilis

Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue ofBacillus subtilis was shown to be induced upon infection with Ø105clz, a clear plaque mutant of the temperate bacteriophage Ø105. Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homologue when cells were infected with Ø105clz.     

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.     

Axial Diffusivity of the Corona Radiata at 24 Hours Post-Stroke: A New Biomarker for Motor and Global Outcome

Fractional anisotropy (FA) is an effective marker of motor outcome at the chronic stage of stroke yet proves to be less efficient at early time points. This study aims to determine which diffusion metric in which location is the best marker of long-term stroke outcome after thrombolysis with diffusion tensor imaging (DTI) at 24 hours post-stroke. Twenty-eight thrombolyzed patients underwent DTI at 24 hours post-stroke onset. Ipsilesional and contralesional FA, mean (MD), axial (AD), and radial (RD) diffusivities values were calculated in different Regions-of-Interest (ROIs): (1) the white matter underlying the precentral gyrus (M1), (2) the corona radiata (CoRad), (3) the posterior limb of the internal capsule (PLIC) and (4) the cerebral peduncles (CP). NIHSS scores were acquired at admission, day 1, and day 7; modified Rankin Scores (mRS) at 3 months. Significant decreases were found in FA, MD, and AD of the ipsilesional CoRad and M1. MD and AD were also significantly lower in the PLIC. The ratio of ipsi and contralesional AD of the CoRad (CoRad-rAD) was the strongest diffusion parameter correlated with motor NIHSS scores on day 7 and with the mRS at 3 months. A Receiver-Operator Curve analysis yielded a model for the CoRad-rAD to predict good outcome based on upper limb NIHSS motor scores and mRS with high specificity and sensitivity. FA values were not correlated with clinical outcome. In conclusion, axial diffusivity of the CoRad from clinical DTI at 24 hours post-stroke is the most appropriate diffusion metric for quantifying stroke damage to predict outcome, suggesting the importance of early axonal damage.     

Use of two formulations and two application techniques to deliver Bacillus thuringiensis var. israelensis for the control of Musca domestica (Diptera: Muscidae) larvae and adults in poultry houses

A field study was carried out for 6 wks to assess, from both an efficiency and economic perspective, the effect of individual and integrated success of feeding and topical applications of two formulations of Bacillus thuringiensis var. israelensis (Bti) in controlling house fly (Musca domestica L.) larvae and adults in poultry houses. There was no significant difference between the 1 g and 2 g L^?1 spray applications of Bti. In the absence of spray applications, no significant differences in larval mortalities were observed between the 250 mg and 500 mg kg^?1 feed applications. The percentage mortality of larvae accomplished as a result of using a combination of 250 mg kg^?1Bti feed and 2 g L^?1 spray applications was equivalent to that obtained as a result of combining 500 mg kg^?1Bti and 1g L^?1 spray applications. Treatment with Bti caused significant reductions in the emergence (up to 74%) of house fly adults compared to the control. The fact that the emergence of adult house flies was affected by Bti treatments implies that Bti has sublethal effects on house fly larvae. The cost–benefit analysis (expressed in terms of mortality of larvae growing) indicated that the most effective combination for house fly larvae and adult house fly emergence control was the 500 mg kg^?1 of feed and 2 g L^?1 spray application combination that resulted in 67% larval mortality and a 74% decrease in adult house fly emergence. This study presents commercial users with various alternatives for possible combinations of the two Bti formulations.     

Clonal abortion of bone marrow T cell precursors: T cells acquire specific antigen reactivity prethymically

Lethally irradiated (900 R) mice were reconstituted with bone marrow cells from syngeneic donors that had been tolerized 2 to 3 wk earlier to either DNP or TNP compounds. Five weeks after reconstitution, these animals were tested for their ability to mount a delayed hypersensitivity (DH) response to the tolerizing haptens. Recipient mice were specifically tolerant to the hapten that was used to induce tolerance in the marrow donor. Mixing experiments in which mice were reconstituted with marrow from DNP-tolerant and TNP-tolerant donors showed no indication of active suppression or effective antigen carry-over in this system. This observation held true even in experiments in which mice were reconstituted with a mixture of marrow from tolerant and normal donors at a ratio of 5:1. Thus the reduced responsiveness in recipient mice seemed to be due to the functional elimination of hapten-responsive T cell precursor (pre-T) clones. Recipient unresponsiveness was also shown to be MHC restricted. Maintenance of unresponsiveness appeared to be due to the restricted access of regenerating pre-T cell clones to the maturational influence of the recipient's thymus.