Combinatorial Assembly of Multigene Pathways by Combining Single-Strand Assembly with Golden Gate Assembly.

Biotechnological production routes for fine and bulk chemicals are progressively explored and developed. Yet this development is hampered by the many constraints determining the metabolic sweet spot, such as optimal expression levels, metabolic stress, feedback regulation, etc.In this regard, we introduce a novel, highly reliable, and rapid single-strand assembly (SSA) methods for combinatorial pathway engineering. In this contribution, SSA is elucidated which enables one to modulate the expression via promoter and/or RBS randomization. Moreover, a new combinatorial multigene pathway assembly scheme based on single-strand assembly (SSA) methods and Golden Gate Assembly is introduced, exploiting the strengths of both assembly techniques.

Proximate causes of intraspecific variation in locomotor performance in the lizard Gallotia galloti.

To understand the evolution of biological traits, information on the degree and origins of intraspecific variation is essential. Because adaptation can take place only if the trait shows heritable variation, it is important to know whether (at least) part of the trait variation is genetically based. We describe intra- and interindividual variation in three performance measures (sprint speed, climbing, and clambering speed) in juvenile Gallotia galloti lizards from three populations and examine how genetic, environmental (incubation temperature), and ontogenetic (age, size) effects interact to cause performance variation. Moreover, we test whether the three performance traits are intercorrelated phenotypically and genetically. Sprint speed is highest in juveniles incubated at the lowest temperature (26 degrees C) irrespective of population. Climbing speed differs among populations, and the differences persist at least until the lizards are 30 wk old. This suggests that the three populations experience different selective pressures. Moreover, mass, snout-vent length, and hindlimb length seem to affect climbing performance differently in the three populations. The variation in sprinting and climbing ability appears to be genetically based. Moreover, the two performance traits are intercorrelated and thus will not evolve independently from each other. Clambering speed (i.e., capacity to climb up an inclined mesh) varies among individuals, but the origin of this variation remains obscure.

Congenital tricuspid valve stenosis with atrial septal defect and left anterior fascicular block.

Congenital tricuspid valve stenosis in the absence of other valvular abnormalities is rare. In this report we describe a patient with congenital tricuspid valve stenosis, ostium secundum atrial septal defect, and electrocardiographic left anterior fascicular block, who presented with paradoxical emboli. This case, as well as previous case reports, suggests that congenital tricuspid stenosis with ostium secundum atrial septal defect is associated with left anterior fascicular block.

Pregnancy and humoral immune response in mice chronically infected by Trypanosoma cruzi.

The effect of pregnancy on the humoral immune response induced by Trypanosoma cruzi was studied in groups of chronically infected and pregnant mice (IP) or chronically infected and nonpregnant mice (INP) of strain BALB/c. Groups of noninfected and nonpregnant mice (NINP) or noninfected and pregnant mice (NIP) served as controls. The pregnant mice were killed on day 17 of pregnancy. Anti-T. cruzi immunoglobulin G (IgG) and IgM antibodies, detected by immunofluorescence or enzyme-linked immunosorbent assay or both, underwent a pregnancy-associated decrease of 20 to 40%, whereas complement-mediated lytic antibodies were unaffected by pregnancy. Immunoblotting analysis indicated identical specificities of the anti-T. cruzi antibodies in IP and INP groups. The levels of all the immunoglobulin isotypes (particularly IgG2a and IgG3), circulating immune complexes, rheumatoid-like factor, and anti-DNA antibodies were considerably increased during chronic infection (NINP versus INP), which could be related to the high degree of polyclonal B-cell activation occurring in T. cruzi infection. However, pregnancy significantly decreased (by 20 to 60%) such parameters. IgG levels were particularly affected (by 40 to 60%), and the decreases could be ordered as follows: IgG3 greater than IgG2a greater than IgG1 greater than IgG2b for IP versus INP. Comparisons between the noninfected groups indicated differences only in IgG levels. These results indicate the following. (i) The specific humoral anti-T. cruzi immune response is weakly affected by pregnancy, which is not sufficient to modify the course of the mother's infection. (ii) Pregnancy does not modify the expression of the anti-T. cruzi antibody repertory. (iii) Pregnancy reduces the polyclonal B-cell activation, particularly the levels of the IgG isotypes undergoing the greatest activation.

Effects of myocardial ischemia on quantitative ultrasonic backscatter and identification of responsible determinants.

Quantitative characterization of myocardial properties represent a rapidly emerging area of echocardiographic investigation. Because measurement of the ultrasonic integrated backscatter is theoretically applicable to analysis in vivo with reflected ultrasound, this study was performed to develop and evaluate a suitable method for measurement of quantitative backscatter in vivo. In view of the desirability of characterizing ischemic myocardium noninvasively, the study was performed with animal preparations simulating myocardial ischemia in humans. In one series of open-chest dogs, integrated backscatter among 22 ischemic regions was increased by 200% (P less than 0.01) compared to values in control regions within 1 hour after coronary occlusion and by 400% (-45.1 +/- 0.7 dB compared to -50.9 +/- 0.4 dB) (P less than 0.001) within 6 hours. In a second series of open-chest dogs, ischemia was quantified with 141Ce microspheres, and mean integrated backscatter was found to increase (280% of control) (P less than 0.01) in regions with flow less than 20% of control 2 hours following coronary occlusion. Additional studies with perfused hearts revealed two determinants of the increased ultrasonic backscatter observed: (1) an increase in cardiac fluid content reflected by the wet-to-dry weight ratio, and (2) the contributions of formed elements in whole blood. The results indicate that ultrasonic integrated backscatter distinguishes severely ischemic from nonischemic myocardium in vivo in open-chest animals. Because it was possible to obtain these results in the reflection mode, potential extension of the approach to clinical applications is promising.