Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.     

Plant exudates promote PCB degradation by a rhodococcal rhizobacteria

Rhodococcus erythropolis U23A is a polychlorinated biphenyl (PCB)-degrading bacterium isolated from the rhizosphere of plants grown on a PCB-contaminated soil. Strain U23A bphA exhibited 99% identity with bphA1 of Rhodococcus globerulus P6. We grew Arabidopsis thaliana in a hydroponic axenic system, collected, and concentrated the plant secondary metabolite-containing root exudates. Strain U23A exhibited a chemotactic response toward these root exudates. In a root colonizing assay, the number of cells of strain U23A associated to the plant roots (5.7?×?10? CFU g?1) was greater than the number remaining in the surrounding sand (4.5?×?10? CFU g?1). Furthermore, the exudates could support the growth of strain U23A. In a resting cell suspension assay, cells grown in a minimal medium containing Arabidopsis root exudates as sole growth substrate were able to metabolize 2,3,4'- and 2,3',4-trichlorobiphenyl. However, no significant degradation of any of congeners was observed for control cells grown on Luria-Bertani medium. Although strain U23A was unable to grow on any of the flavonoids identified in root exudates, biphenyl-induced cells metabolized flavanone, one of the major root exudate components. In addition, when used as co-substrate with sodium acetate, flavanone was as efficient as biphenyl to induce the biphenyl catabolic pathway of strain U23A. Together, these data provide supporting evidence that some rhodococci can live in soil in close association with plant roots and that root exudates can support their growth and trigger their PCB-degrading ability. This suggests that, like the flagellated Gram-negative bacteria, non-flagellated rhodococci may also play a key role in the degradation of persistent pollutants.     

Effects of chlorobenzoate transformation on the Pseudomonas testosteroni biphenyl and chlorobiphenyl degradation pathway.

Bacterial conversion of biphenyl (BP) and chlorobiphenyls (CBPs) to benzoates and chlorobenzoates (CBAs) proceeds by introduction of molecular oxygen at the 2,3 position, followed by a 1,2-meta cleavage of the molecule. Complete mineralization of CBPs requires the presence of two sets of genes, one for the transformation fo CBPs into CBAs and a second for the degradation of CBAs. It has been shown previously that removal of the CBAs produced from the degradation of CBPs is essential for efficient degradation of CBPs. In this study we confirmed that CBAs inhibit BP and CBP transformation in Pseudomonas testosteroni B-356. Among the three monochlorobenzoates tested, 3-chlorobenzoate was the most effective inhibitor. Furthermore, we found that in strain B-356, CBA transformation is controlled by BP-induced oxygenases that are not present in benzoate-grown cells. We found that this BP-linked CBA transformation pathway transformed CBAs produced from CBPs into several metabolites, including chlorocatechols and corresponding muconic semialdehydes. These metabolites inhibited the 2,3-dihydroxybiphenyl 1,2-dioxygenase, while CBAs by themselves had no effect on this enzyme. Therefore, on the basis of this and other observations, it appears that when CBAs produced from CBPs accumulate in the growth medium, they are converted into unproductive metabolites that reduce the flux of the BP and CBP degradation pathway. The practical implications of these interactions on the microbial degradation of polychlorinated BPs are also discussed.     

Arterial Hypertension and Ischemic Heart Disease: Comparison in Epidemiological Studies

Forty cases of gonococcal urethritis were treated with oxytetracycline using various dosage schedules; there were 37 cures and three failures. The most convenient and most effective dosage was found to be 250 mg. oxytetracycline, given as a single intramuscular injection of 5 c.c.A series of 40 patients with non-gono-coccal urethritis was also collected. Two cases of urethritis due to Trichomonas vaginalis and two due to Candida albicans were removed from the series. Of the 36 cases which remained, cure was obtained with the use of oxytetracycline in different dosages in 30 cases; six cases were failures. The dosage which gave the best result in the therapy of non-gonococcal urethritis was 250 mg. oxytetracycline (5 c.c.), given as a single intramuscular injection, plus 250 mg. orally, four times a day for four days.The effectiveness of oxytetracycline in the treatment of urethritis has not decreased.     

Solving the multifunctionality dilemma in biorefineries with a novel hybrid mass–energy allocation method

Processing biomass into multifunctional products can contribute to food, feed, and energy security while also mitigating climate change. However, biorefinery products nevertheless impact the environment, and this influence needs to be properly assessed to minimize the burden. Life cycle assessment (LCA) is often used to calculate environmental footprints of products, but distributing the burdens among the different biorefinery products is a challenge. A particular complexity arises when the outputs are a combination of energy carrying no mass, and mass carrying no energy, where neither an allocation based on mass nor on energy would be appropriate. A novel hybrid mass–energy (HMEN) allocation scheme for dealing with multifunctionality problems in biorefineries was developed and applied to five biorefinery concepts. The results were compared to results of other allocation methods in LCA. The reductions in energy use and GHG emissions from using the biorefinery's biofuels were also quantified. HMEN fairly distributed impacts among biorefinery products and did not change the order of the products in terms of the level of the pollution caused. The allocation factors for HMEN fell between mass and economic allocation factors and were comparable to energy allocation factors. Where the mass or the energy allocation failed to attribute burdens, HMEN addressed this shortcoming by assigning impacts to nonmass or to nonenergy products. Under the partitioning methods and regardless of the feedstock used, bioethanol reduced GHG by 72–98% relative to gasoline. The GHG savings were 196% under the substitution method, but no GHG savings occurred for sugar beet bioethanol under the surplus method. Bioethanol from cellulosic crops had lower energy use and GHG emissions than from sugar beet, regardless of the allocation method used. HMEN solves multifunctional problems in biorefineries and can be applied to other complex refinery systems. LCA practitioners are encouraged to further test this method in other case studies.     

Pilot study using 3D–longitudinal strain computation in a multi-parametric approach for best selecting responders to cardiac resynchronization therapy

Background

Almost all attempts to improve patient selection for cardiac resynchronization therapy (CRT) using echo-derived indices have failed so far. We sought to assess: the performance of homemade software for the automatic quantification of integral 3D regional longitudinal strain curves exploring left ventricular (LV) mechanics and the potential value of this tool to predict CRT response.

Methods

Forty-eight heart failure patients in sinus rhythm, referred for CRT-implantation (mean age: 65 years; LV-ejection fraction: 26%; QRS-duration: 160 milliseconds) were prospectively explored. Thirty-four patients (71%) had positive responses, defined as an LV end-systolic volume decrease ≥15% at 6-months. 3D–longitudinal strain curves were exported for analysis using custom-made algorithms. The integrals of the longitudinal strain signals (I _L,peak) were automatically measured and calculated for all 17 LV-segments.

Results

The standard deviation of longitudinal strain peak (SDI _L,peak ) for all 17 LV-segments was greater in CRT responders than non-responders (1.18% s^?1 [0.96; 1.35] versus 0.83% s^?1 [0.55; 0.99], p = 0.007). The optimal cut-off value of SDI _L,peak to predict response was 1.037%.s^?1. In the 18-patients without septal flash, SDI _L,peak was significantly higher in the CRT-responders.

Conclusions

This new automatic software for analyzing 3D longitudinal strain curves is avoiding previous limitations of imaging techniques for assessing dyssynchrony and then its value will have to be tested in a large group of patients.

     

Structure of the OmpA-like domain of RmpM from Neisseria meningitidis

RmpM is a putative peptidoglycan binding protein from Neisseria meningitidis that has been shown to interact with integral outer membrane proteins such as porins and TonB-dependent transporters. Here we report the 1.9 A crystal structure of the C-terminal domain of RmpM. The 150-residue domain adopts a betaalphabetaalphabetabeta fold, as first identified in Bacillus subtilis chorismate mutase. The C-terminal RmpM domain is homologous to the periplasmic, C-terminal domain of Escherichia coli OmpA; these domains are thought to be responsible for non-covalent interactions with peptidoglycan. From the structure of the OmpA-like domain of RmpM, we suggest a putative peptidoglycan binding site and identify residues that may be essential for binding. Both the crystal structure and solution experiments indicate that RmpM may exist as a dimer. This would promote more efficient peptidoglycan binding, by allowing RmpM to interact simultaneously with two glycan chains through its C-terminal, OmpA-like binding domain, while its (structurally uncharacterized) N-terminal domain could stabilize oligomers of porins and TonB-dependent transporters in the outer membrane.     

Carbohydrate Production in Relation to Microphytobenthic Biofilm Development: An Integrated Approach in a Tidal Mesocosm

Experiments were performed to evaluate short-term changes in sediment extracellular carbohydrates for a multispecific assemblage of benthic diatoms in relation to physiological status, endogenous migratory rhythms, and environmental conditions. For this purpose, a mesocosm was used, which simulated both tidal and dark: light alternating cycles under controlled conditions. Scanning electronic microscopy in combination with picture analyses indicated that natural diatom migration patterns were reproduced in the mesocosm. Two EPS fractions were operationally separated in colloidal carbohydrate measurements: alcohol-soluble EPS (termed “soluble EPS”) and alcohol-insoluble EPS (termed “bound EPS”). Microphytobenthic biomass followed a logistic-type curve and converged toward a maximal value termed the “biotic capacity of the local environment.” Both EPS fractions showed oscillations with production during photosynthetic periods and sharp decreases during night immersion periods. Productions of both EPS fractions increased with Chl a production during light periods suggesting a light dependence in relation to migratory patterns. The decreases in both EPS fractions, which occurred during night immersion periods suggest that carbohydrate hydrolysis and/or washaway affected both EPS fractions similarly in benthic environments. Our results confirm the theory according to which the two distinct fractions are under different metabolic controls. No change in soluble EPS release was obtained during the transition from logarithmic to stationary phase. On the other hand, a metabolism modification of microalgae, probably related to ammonium depletion, occurred when cells entered the stationary phase, since there was a high enhancement in bound EPS production. Mesocosm results can serve as a system of reference useful to characterize biofilm development in field investigations and to revisit the effective implication of each EPS fraction in sediment stability.