Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Some effects of tetracaine on rat heart mitochondria

The effect of the local anesthetic tetracaine on some functions of rat heart mitochondria was studied, and the results show that this tertiary amine inhibits the oxidation of NAD-dependent substrates by affecting the mitochondrial membrane in a site located between the NADH dehydrogenase and Co Q span through a process which is not affected by K⁺, Ca2⁺, or Mg2⁺. Also the results show that tetracaine induces sodium uptake probably by a H⁺/Na⁺ exchange reaction.     

Only the Carrot,Not the Stick: Incorporating Trust into the Enforcement of Regulation

New enforcement strategies allow agents to gain the regulator’s trust and consequently face a lower audit probability. Prior research suggests that, in order to prevent lower compliance, a reduction in the audit probability (the “carrot”) must be compensated with the introduction of a higher penalty for non-compliance (the “stick”). However, such carrot-and-stick strategies reflect neither the concept of trust nor the strategies observed in practice. In response to this, we define trust-based regulation as a strategy that incorporates rules that allow trust to develop, and using a generic (non-cooperative) game of tax compliance, we examine whether trust-based regulation is feasible (i.e., whether, in equilibrium, a reduction in the audit probability, without ever increasing the penalty for non-compliance, does not lead to reduced compliance). The model shows that trust-based regulation is feasible when the agent sufficiently values the future. In line with the concept of trust, this strategy is feasible when the regulator is uncertain about the agent’s intentions. Moreover, the model shows that (i) introducing higher penalties makes trust-based regulation less feasible, and (ii) combining trust and forgiveness can lead to a lower audit probability for both trusted and distrusted agents. Policy recommendations often point toward increasing deterrence. This model shows that the opposite can be optimal.     

Nonlinear viscoelastic constitutive model for bovine liver tissue

Soft tissue mechanical characterisation is important in many areas of medical research. Examples span from surgery training, device design and testing, sudden injury and disease diagnosis. The liver is of particular interest, as it is the most commonly injured organ in frontal and side motor vehicle crashes, and also assessed for inflammation and fibrosis in chronic liver diseases. Hence, an extensive rheological characterisation of liver tissue would contribute to advancements in these areas, which are dependent upon underlying biomechanical models. The aim of this paper is to define a liver constitutive equation that is able to characterise the nonlinear viscoelastic behaviour of liver tissue under a range of deformations and frequencies. The tissue response to large amplitude oscillatory shear (1–50%) under varying preloads (1–20%) and frequencies (0.5–2 Hz) is modelled using viscoelastic-adapted forms of the Mooney–Rivlin, Ogden and exponential models. These models are fit to the data using classical or modified objective norms. The results show that all three models are suitable for capturing the initial nonlinear regime, with the latter two being capable of capturing, simultaneously, the whole deformation range tested. The work presented here provides a comprehensive analysis across several material models and norms, leading to an identifiable constitutive equation that describes the nonlinear viscoelastic behaviour of the liver.

     

Lipophilic 9,10‐Dehydrofukinone Action on Pathogenic and Non‐Pathogenic Bacterial Biofilms. Why Is This Main Volatile Metabolite in Senecio?

The effect of a natural sesquiterpene ketone, 9,10‐dehydrofukinone (DHF), on pathogenic Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from chronic infectious processes, was the focus of the present study. Lipophilic DHF produced important antibacterial synergistic effects in association with ciprofloxacin (CPX) against two biofilm‐forming strains of S. aureus HT1 (FIC=0.21) and P. aeruginosa HT5 (FIC=0.05). Hence, this mixture constitutes an excellent strategy to combat these biofilm‐producing bacteria that overexpress drug efflux pumps as a resistance mechanism. Additionally, a substantial rise in beneficial Lactobacillus biofilm biomass was determined as a very significant finding of this association. Particularly, a non‐pathogenic biofilm increment of 119 % was quantified when the mixture was added to a probiotic L. acidophilus ATCC SD‐5212 culture. A surface activity enhanced in 71 % with respect to untreated L. acidophilus culture was also generated by the DHF and CPX association, and therefore, a glycoprotein synthesis induction mediated by the mixture is discussed. The results obtained could help in the development of new selective antibiotics. From an ecological standpoint, the present study strongly suggests that DHF is a polyfunctional organic molecule produced with a high yield in Senecio punae that exerts a positive impact on a non‐pathogenic plant bacterium L. plantarum CE105.     

Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.     

Role of a noncanonical disulfide bond in the stability,affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB

A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (T_m = 12°C lower than wild‐type), and a loss of the ability to fold reversibly due to heat induced aggregation. X‐ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B‐factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.     

Assessing life cycle environmental impacts of inoculating soybeans in Argentina with Bradyrhizobium japonicum

The International Journal of Life Cycle Assessment - To estimate life cycle impacts from introducing the yield-enhancing inoculant containing the nitrogen-fixing bacterium Bradyrhizobium japonicum...     

Genetic diversity in oak populations under intensive management for fuelwood in the Sierra de Zongolica,Mexico

Firewood and charcoal are used on a daily basis both in rural areas and in cities. This type of energy is produced by one of the most ancient traditional methods, known as coppice, which harvest tree sprouts. There is controversy about its effects on forests: it preserves populations and tree cover of species used, but reduces density, inhibits sexual reproduction and generates genetic erosion. We inquired if it was possible to identify a loss of genetic diversity in oak populations traditionally used for charcoal by the Zongolica Nahuas in Veracruz state, Mexico. We studied populations of Quercus laurina, Quercus calophylla and Quercus rugosa in three different altitudes. Molecular analysis with eight nuclear codominant microsatellites was performed to determine the diversity, structure and gene flow of these species. Results for Q. laurina were Na = 8.458, I = 1.766, Ho = 0.679, polymorphism = 100%, Fis = 0.079, with intraindividual variation of 81.55%. For Q. calophylla: Na = 7.250, I = 1.563, Ho = 0.646, polymorphism = 91.67%, Fis = 0.083, with intraindividual variation of 83.80%. For Q. rugosa: Na = 6.958, I = 1.510, Ho = 0.574, polymorphism = 91.67%, Fis = 0.204, with intraindividual variation of 81.99%; this species shows signals of an early genetic isolation process. Our findings indicate that Quercus genetic diversity for the three species is high and comparable with oak species in Mexico and worldwide. We conclude that at the present, coppice is preserving a historical diversity in adult trees kept alive through sprouting. Nonetheless, problems with coppice systems elsewhere, unregulated harvesting and expansion of pine plantation in the region suggest that further studies, hand in hand with a landscape management approach that improve charcoal and firewood production, may be valuable for Sierra de Zongolica genetic biodiversity conservation.