Core temperature measurement by microwave radiometry

A ‘double-container model’ was used for core temperature (T_c) measurement by microwave emission radiometry (MR) of warm fluid inside a tube, placed in a container with a cooler fluid. The intensity of microwaves emitted from the warmer fluid inside the tube were measured using a MR metering device, consisting of an antenna linked to a low-noise radio frequency amplifier (bandwidth 500 MHz, centered at 4.0 GHz). Based on the MR measurements, a T_c prediction model was developed for measuring the temperature of fluid inside a tube, achieving a sensitivity of ±0.5 °C at environmental temperature of 33–37 °C.     

The structural basis of the thermostability of SP1, a novel plant (Populus tremula) boiling stable protein

We previously reported on a new boiling stable protein isolated from aspen plants (Populus tremula), which we named SP1. SP1 is a stress-related protein with no significant sequence homology to other stress-related proteins. It is a 108-amino-acid hydrophilic polypeptide with a molecular mass of 12.4 kDa (Wang, W. X., Pelah, D., Alergand, T., Shoseyov, O., and Altman, A. (2002) Plant Physiol. 130, 865-875) and is found in an oligomeric form. Preliminary electron microscopy studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that SP1 is a dodecamer composed of two stacking hexamers. We performed a SDS-PAGE analysis, a differential scanning calorimetric study, and crystal structure determination to further characterize SP1. SDS-PAGE indicated a spontaneous assembly of SP1 to one stable oligomeric form, a dodecamer. Differential scanning calorimetric showed that SP1 has high thermostability i.e. Tm of 107 degrees C (at pH 7.8). The crystal structure of SP1 was initially determined to 2.4 A resolution by multi-wavelength anomalous dispersion method from a crystal belonging to the space group I422. The phases were extended to 1.8 A resolution using data from a different crystal form (P21). The final refined molecule includes 106 of the 108 residues and 132 water molecules (on average for each chain). The R-free is 20.1%. The crystal structure indicated that the SP1 molecule has a ferredoxin-like fold. Strong interactions between each two molecules create a stable dimer. Six dimers associate to form a ring-like-shaped dodecamer strongly resembling the particle visualized in the electron microscopy studies. No structural similarity was found between the crystal structure of SP1 and the crystal structure of other stress-related proteins such as small heat shock proteins, whose structure has been already determined. This structural study further supports our previous report that SP1 may represent a new family of stress-related proteins with high thermostability and oligomerization.     

Improving Biocatalyst Performance by Integrating Statistical Methods into Protein Engineering

Directed evolution and rational design were used to generate active variants of toluene-4-monooxygenase (T4MO) on 2-phenylethanol (PEA), with the aim of producing hydroxytyrosol, a potent antioxidant. Due to the complexity of the enzymatic system—four proteins encoded by six genes—mutagenesis is labor-intensive and time-consuming. Therefore, the statistical model of Nov and Wein (J. Comput. Biol. 12:247-282) was used to reduce the number of variants produced and evaluated in a lab. From an initial data set of 24 variants, with mutations at nine positions, seven double or triple mutants were identified through statistical analysis. The average activity of these mutants was 4.6-fold higher than the average activity of the initial data set. In an attempt to further improve the enzyme activity to obtain PEA hydroxylation, a second round of statistical analysis was performed. Nine variants were considered, with 3, 4, and 5 point mutations. The average activity of the variants obtained in the second statistical round was 1.6-fold higher than in the first round and 7.3-fold higher than that of the initial data set. The best variant discovered, TmoA I100A E214G D285Q, exhibited an initial oxidation rate of 4.4 ± 0.3 nmol/min/mg protein, which is 190-fold higher than the rate obtained by the wild type. This rate was also 2.6-fold higher than the activity of the wild type on the natural substrate toluene. By considering only 16 preselected mutants (out of ∼13,000 possible combinations), a highly active variant was discovered with minimum time and effort.Enzymes are remarkable biocatalysts that perform numerous chemical reactions. They have evolved in nature to do their task in an efficient and specific way, mostly under aqueous physiological conditions (12). However, the term “biocatalysis” refers to the use of enzymes as process catalysts under artificial conditions, and a major challenge today is to render biocatalysts suitable for the tough reaction conditions of an industrial process (11).A widely used approach for improving enzyme function is directed evolution, whereby protein sequences are repeatedly selected, mutated, or recombined in a process that mimics natural evolution to produce better and better “generations” of protein variants. Directed evolution has been successfully used in numerous studies, but since it requires generation, purification, and screening of large numbers of variants, it is typically expensive and labor-intensive (28, 35). An alternative to directed evolution is an approach termed rational design, whereby predictions are made as to how mutations in a protein will affect its structure and hence its interaction with the target molecule. Unfortunately, both the sequence-structure and the structure-activity relationships are extremely intricate, and while this approach proved to be fruitful in some cases, its practical use is still limited (15). The rational-design approach also requires knowledge of the three-dimensional structure of the protein, which, unlike with the protein''s sequence, is costly and time-consuming to decipher. It has been suggested lately that a combination of both methods may be the best tactic to obtain enzymes with desired activities and selectivities (15, 23).Yet a third approach for protein improvement, which is less often used, is based on statistical analysis. According to this approach, the activity of any protein variant is viewed as a random quantity, and statistical methods are used to predict from activity data which mutation combinations are likely to improve activity. The statistical approach does not require structural knowledge about the protein at hand and allows one to focus screening efforts on a few promising variants, thus reducing labor, time, and expenses. Earlier statistical models for the sequence-activity relationship include Kauffman''s NK model (14) and the “rough Mt. Fuji” model of Aita and Husimi (1). More recently, Fox et al. combined a machine learning technique termed ProSAR with directed evolution and rational design to significantly increase the catalytic function of a halohydrin dehalogenase in the production of the cholesterol-lowering drug atorvastatin (Lipitor) (9, 10). Liao et al. (18) employed eight machine learning algorithms to improve 20-fold the ability of proteinase K to hydrolyze a tetrapeptide substrate.The statistical model that lies at the center of this work is that of Nov and Wein (22). Unlike the statistical algorithms used in the work of Fox et al. (9, 10) and Liao et al. (18), which are generic methods from the machine learning literature, this model was devised specifically for the protein design problem to capture characteristics of the protein sequence-activity relationship. Barak et al. (3) employed a variation of this model in conjunction with directed evolution to greatly improve the oxidoreductase ChrR in reducing chromate and uranyl.In this work, we combine all three approaches—directed evolution, rational design, and statistical methods—to improve the capacity of toluene 4-monooxygenase (T4MO) to produce an important antioxidant, hydroxytyrosol (6). This phenol, which is naturally present in olives and olive oil, has the highest free radical scavenging capacity and has been shown to be beneficial in preventing various diseases, such as diabetes, atherosclerosis, and cancer (13, 19, 34). Developing a biotechnological process for the synthesis of this antioxidant is of interest to the food and cosmetics industries.T4MO from Pseudomonas mendocina KR1 is a soluble four-component enzyme belonging to the toluene monooxygenase family. T4MO is composed of six genes, designated tmoABCDEF, which are essential for the efficient catalysis and high regiospecificity of the enzyme. Genes tmoA, tmoB, and tmoE encode the α, β, and γ subunits, respectively, that comprise the (αβγ)₂ quaternary structure of the 212-kDa hydroxylase component. The α hydroxylase subunit contains the catalytically active diiron center (2, 8). The tmoC, tmoD, and tmoF genes encode the 12.5-kDa Rieske-type [2Fe-2S] ferredoxin, the 11.6-kDa effector protein, and the 36-kDa NADH oxidoreductase, respectively (2, 16, 21).Previously, a 35-fold improvement in T4MO activity on 2-phenylethanol (PEA) for the synthesis of hydroxytyrosol was reported for the TmoA I100A mutant (5). The goal of the present work was to further generate a better T4MO variant for the production of hydroxytyrosol. As the cloning steps associated with producing double and triple mutants of this enzyme are very laborious and time-consuming (e.g., QuikChange mutagenesis cannot be applied due to the large plasmid involved [9 kb], and a three-step PCR is needed for each mutant [5, 6]), the integration of the Nov and Wein statistical model was evaluated.     

Identification and Localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor “Candidatus Portiera aleyrodidarum,” an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling

SPINDLY (SPY) is a negative regulator of gibberellin (GA) responses; however, spy mutants exhibit various phenotypic alterations not found in GA-treated plants. Assaying for additional roles for SPY revealed that spy mutants are resistant to exogenously applied cytokinin. GA also repressed the effects of cytokinin, suggesting that there is cross talk between the two hormone-response pathways, which may involve SPY function. Two spy alleles showing severe (spy-4) and mild (spy-3) GA-associated phenotypes exhibited similar resistance to cytokinin, suggesting that SPY enhances cytokinin responses and inhibits GA signaling through distinct mechanisms. GA and spy repressed numerous cytokinin responses, from seedling development to senescence, indicating that cross talk occurs early in the cytokinin-signaling pathway. Because GA3 and spy-4 inhibited induction of the cytokinin primary-response gene, type-A Arabidopsis response regulator 5, SPY may interact with and modify elements from the phosphorelay cascade of the cytokinin signal transduction pathway. Cytokinin, on the other hand, had no effect on GA biosynthesis or responses. Our results demonstrate that SPY acts as both a repressor of GA responses and a positive regulator of cytokinin signaling. Hence, SPY may play a central role in the regulation of GA/cytokinin cross talk during plant development.     

Effects of floral traits and plant genetic composition on pollinator behavior

Pollinator-mediated selection plays a major role in floral evolution and speciation. Floral traits that influence animal pollinator behavior are the target of pollinator-mediated selection, but can only evolve if floral phenotypes have underlying genetic variation. Thus, understanding the genetic basis of a floral trait is a crucial step in studying pollinator-mediated selection. In this study I tested the effect of quantitative trait loci (QTL) underlying floral traits on pollinator behavior in recombinant inbred lines (RILs) in the common sunflower, Helianthus annuus L. and its crop relative. The indirect effects of QTL on pollinator behavior, mediated by floral phenotypes, were analyzed for six insect visitor types using structural equation modeling (SEM) and path analysis. For three of the six visitor types (large and small bees and non-bee insects) valid models were revealed when all three levels (QTL, floral traits, and pollinator behavior) were incorporated. Nested model without genetics were validated for five of the six visitor types. The results suggest that insect behavior as a reaction to floral phenotypes is affected by the genetic architecture of floral traits. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: Heikki Hokkanen