Rational mutagenesis of Thermobifida fusca cutinase to modulate the enzymatic degradation of polyethylene terephthalate

Thermobifida fusca cutinase (TfCut2) is a carboxylesterase (CE) which degrades polyethylene terephthalate (PET) as well as its degradation intermediates [such as oligoethylene terephthalate (OET), or bis-/mono-hydroxyethyl terephthalate (BHET/MHET)] into terephthalic acid (TPA). Comparisons of the surfaces of certain CEs (including TfCut2) were combined with docking and molecular dynamics simulations involving 2HE-(MHET)_3, a three-terephthalate OET, to support the rational design of 22 variants with potential for improved generation of TPA from PET, comprising 15 single mutants (D12L, E47F, G62A, L90A, L90F, H129W, W155F, ΔV164, A173C, H184A, H184S, F209S, F209I, F249A, and F249R), 6 double mutants [H129W/T136S, A173C/A206C, A173C/A210C, G62A/L90F, G62A/F209I, and G62A/F249R], and 1 triple mutant [G62A/F209I/F249R]. Of these, nine displayed no activity, three displayed decreased activity, three displayed comparable activity, and seven displayed increased (~1.3- to ~7.2-fold) activity against solid PET, while all variants displayed activity against BHET. Of the variants that displayed increased activity against PET, four displayed more activity than G62A, the most-active mutant of TfCut2 known till date. Of these four, three displayed even more activity than LCC (G62A/F209I, G62A/F249R, and G62A/F209I/F249R), a CE known to be ~5-fold more active than wild-type TfCut2. These improvements derived from changes in PET binding and not changes in catalytic efficiency.     

Propulsion of a fin whale (Balaenoptera physalus): why the fin whale is a fast swimmer

Measurements of an immature fin whale (Balaenoptera physalus), which died as a result of entrapment in fishing gear near Frenchmans Cove, Newfoundland (47 degrees 9' N, 55 degrees 25' W), were made to obtain estimates of volume and surface area of the animal. Detailed measurements of the flukes, both planform and sections, were also obtained. A strip theory was developed to calculate the hydrodynamic performance of the whale's flukes as an oscillating propeller. This method is based on linear, two-dimensional, small-amplitude, unsteady hydrofoil theory with correction factors used to account for the effects of finite span and finite amplitude motion. These correction factors were developed from theoretical results of large-amplitude heaving motion and unsteady lifting-surface theory. A model that makes an estimate of the effects of viscous flow on propeller performance was superimposed on the potential-flow results. This model estimates the drag of the hydrofoil sections by assuming that the drag is similar to that of a hydrofoil section in steady flow. The performance characteristics of the flukes of the fin whale were estimated by using this method. The effects of the different correction factors, and of the frictional drag of the fluke sections, are emphasized. Frictional effects in particular were found to reduce the hydrodynamic efficiency of the flukes significantly. The results are discussed and compared with the known characteristics of fin-whale swimming.     

Functional dissection of Timekeeper (Tik) implicates opposite roles for CK2 and PP2A during Drosophila neurogenesis

Repression by E(spl)M8 during inhibitory Notch (N) signaling (lateral inhibition) is regulated, in part, by protein kinase CK2, but the involvement of a phosphatase has been unclear. The studies we report here employ Tik, a unique dominant‐negative (DN) mutation in the catalytic subunit of CK2, in a Gal4‐UAS based assay for impaired lateral inhibition. Specifically, overexpression of Tik elicits ectopic bristles in N⁺ flies and suppresses the retinal defects of the gain‐of‐function allele N^spl. Functional dissection of the two substitutions in Tik (M¹⁶¹K and E¹⁶⁵D), suggests that both mutations contribute to its DN effects. While the former replacement compromises CK2 activity by impairing ATP‐binding, the latter affects a conserved motif implicated in binding the phosphatase PP2A. Accordingly, overexpression of microtubule star (mts), the PP2A catalytic subunit closely mimics the phenotypic effects of loss of CK2 functions in N⁺ or N^spl flies, and elicits notched wings, a characteristic of N mutations. Our findings suggest antagonistic roles for CK2 and PP2A during inhibitory N signaling. genesis 47:647–658, 2009. © 2009 Wiley‐Liss, Inc.     

An Ecological Risk Assessment Methodology for Screening Discharge Alternatives of Produced Water

Previous studies on Ecological Risk Assessment (ERA) of produced water relied on the use of deterministic hydrodynamic models. The assessment was usually carried out in the North Sea context using a model such as the Chemical Hazard Assessment and Risk Management (CHARM), or in the North American context based on the output of a hydrodynamic model such as the Cornell Mixing Zone Expert System (CORMIX). In both these cases, however, probabilistic analysis has not been employed, particularly, to account for uncertainty associated with hydrodynamic models in the ERA study. In fact, it is the hydrodynamic model that has a direct linkage to the selection of the discharge alternatives. Apart from the monitoring purposes, in this article, it is suggested that criteria for evaluating discharge alternatives of produced water in a marine environment might incorporate an awareness of ecological risks by incorporating engineering and toxicological aspects. An ERA methodology consisting of problem formulation, analysis, and risk characterization is discussed in light of evaluating the discharge alternatives. A probabilistic analysis using Latin Hypercube Sampling (LHS)–based Monte Carlo (MC) simulations was employed. A depiction of associated risks for an area comparable to a regulatory mixing zone of typical effluent discharges is presented.     

Role of the ribosome in protein folding

In all organisms, the ribosome synthesizes and folds full length polypeptide chains into active three-dimensional conformations. The nascent protein goes through two major interactions, first with the ribosome which synthesizes the polypeptide chain and holds it for a considerable length of time, and then with the chaperones. Some of the chaperones are found in solution as well as associated to the ribosome. A number of in vitro and in vivo experiments revealed that the nascent protein folds through specific interactions of some amino acids with the nucleotides in the peptidyl transferase center (PTC) in the large ribosomal subunit. The mechanism of this folding differs from self-folding. In this article, we highlight the folding of nascent proteins on the ribosome and the influence of chaperones etc. on protein folding.     

Subcutaneous adiposity and relative fat patterning in adult white and migrant asian males in Britain

A comparative study of subcutaneous adiposity and relative fat patterning in adult White (n=262) and migrant Indian (n=39) and Pakistani (n=100) males living in Peterborough, Cambridgeshire revealed no significant difference in the level of generalised adiposity (measured as body mass index) between the ethnic groups. However, Asians had significantly higher means for all five truncal skinfolds; for all upper body: upper extremity, upper body: lower extremity, central body: upper extremity and central body: lower extremity skinfold rations; more total subcutaneous adiposity; and significantly more subcutaneous fat in subscapular, suprailiac and abdomen regions relative to total subcutaneous fat. However, Asian men had significantly less subcutaneous fat in all the extremity sites relative to total subcutaneous adiposity and lower mean forearm subcutaneous adiposity. Discriminant analysis revealed that 80.6% of all individuals were correctly classfied, with Whites being grouped more correctly than Asians. Suprailiac/forearm and suprailiac/triceps skinfold ratios, height, weight, age, body mass index, and subscapular, midauxillary, chest and medial calf skinfolds were among the most important discriminating variables/ratios of the three ethnic groups.     

Alterations in transmission, vesicle dynamics, and transmitter release machinery at NCAM-deficient neuromuscular junctions.

Although functional neuromuscular junctions (NMJs) form in NCAM-deficient mice, they exhibit multiple alterations in presynaptic organization and function. Profound depression and unusual periodic total transmission failures with repetitive stimulation point to a defect in vesicle mobilization/cycling, and these defects were mimicked in (+/+) NMJs by inhibitors of myosin light chain kinase, known to affect vesicle mobilization. Two separate release mechanisms, utilizing different endocytic machinery and Ca(2+) channels, were shown to coexist in (-/-) terminals, with the mature process targeted to presynaptic membrane opposed to muscle, and an abnormally retained immature process targeted to the remainder of the presynaptic terminal and axon. Thus, NCAM plays a critical and heretofore unsuspected role in the molecular organization of the presynaptic NMJ.     

B-box protein BBX32 integrates light and brassinosteroid signals to inhibit cotyledon opening

Cotyledon opening is a key morphological change that occurs in seedlings during de-etiolation. Brassinosteroids (BRs) inhibit the opening of cotyledons in darkness while light promotes cotyledon opening. The molecular regulation of the interplay between light and BR to regulate cotyledon opening is not well understood. Here, we show the B-box protein BBX32 negatively regulates light signaling and promotes BR signaling to inhibit cotyledon opening in Arabidopsis (Arabidopsis thaliana). BBX32 is highly expressed in the cotyledons of seedlings during de-etiolation. bbx32 and 35S:BBX32 seedlings exhibit enhanced and reduced cotyledon opening, respectively, in response to both light and brassinazole treatment in dark, suggesting that BBX32 mediates cotyledon opening through both light and BR signaling pathways. BBX32 expression is induced by exogenous BR and is upregulated in bzr1-1D (BRASSINAZOLE RESISTANT1-1D). Our in vitro and in vivo interaction studies suggest that BBX32 physically interacts with BZR1. Further, we found that PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) interacts with BBX32 and promotes BR-mediated cotyledon closure. BBX32, BZR1, and PIF3 regulate the expression of common target genes that modulate the opening and closing of cotyledons. Our work suggests BBX32 integrates light and BR signals to regulate cotyledon opening during de-etiolation.