Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

Temporal fatty acid dynamics of the octocoral Veretillum cynomorium

The objectives of the present work were to investigate the temporal variation in the fatty acid (FA) composition of the octocoral Veretillum cynomorium, examine the effects of reproduction and environmental factors on FA variation, and establish a chemotaxonomic identification for this species. Mean oocyte size-frequency distributions showed that the majority of the oocytes had an intermediate size (Group II) before spawning (April and June). The late-vitellogenic oocytes (Group III) became absent in August and October and, during this post-spawning period, oocytes were primarily of small size (Group I). Most of the major FA, 16:0, 18:0, 20:4n-6, 20:5n-3, and the tetracosapolyenoic fatty acid (TPA), 24:6n-3, varied significantly throughout the year (p < 0.01), with two peaks in August/October and February. The boost in early oogenesis, also associated with warmer temperatures, seemed to be responsible for the observed increase in FA content between June and August. The highest values of FA content were observed in February when intermediate oogenesis (Group II) was at its peak and there were considerable levels of available food in the environment. Also, the increase in food availability seemed to trigger the final stages of gametogenesis. The high quantity of 18:1n-7, odd-numbered and branched FAs, suggested the presence of a dynamic bacterial community in V. cynomorium, probably as an adaptive response to the lack of symbiotic microalgae. Although the presence of TPAs is the main feature distinguishing octocorals from other coral species, here we showed that there was no single FA clearly dominating the FA composition of V. cynomorium throughout the year. Instead, four main FAs share similar concentrations: 16:0, 20:4n-6, 20:5n-3 and 24:6n-3. The predominance of these four FAs combined with the higher amount of 24:6n-3 when compared to 24:5n-6 may serve as a chemotaxonomic feature to distinguish this octocoral species (or genus).     

CENP-V is required for centromere organization, chromosome alignment and cytokinesis

The mechanism of mitotic chromosome condensation is poorly understood, but even less is known about the mechanism of formation of the primary constriction, or centromere. A proteomic analysis of mitotic chromosome scaffolds led to the identification of CENP-V, a novel kinetochore protein related to a bacterial enzyme that detoxifies formaldehyde, a by-product of histone demethylation in eukaryotic cells. Overexpression of CENP-V leads to hypercondensation of pericentromeric heterochromatin, a phenotype that is abolished by mutations in the putative catalytic site. CENP-V depletion in HeLa cells leads to abnormal expansion of the primary constriction of mitotic chromosomes, mislocalization and destabilization of the chromosomal passenger complex (CPC) and alterations in the distribution of H3K9me3 in interphase nucleoplasm. CENP-V-depleted cells suffer defects in chromosome alignment in metaphase, lagging chromosomes in anaphase, failure of cytokinesis and rapid cell death. CENP-V provides a novel link between centromeric chromatin, the primary constriction and the CPC.     

Redox Properties of Thermus thermophilus ba3: Different Electron-Proton Coupling in Oxygen Reductases?

A comprehensive study of the thermodynamic redox behavior of the hemes of the ba₃ enzyme from Thermus thermophilus, a B-type heme-copper oxygen reductase, is presented. This enzyme, in contrast to those having a single type of heme, allows the B- and A-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. The relative order of the midpoint reduction potentials of each center changed in the pH range from 6 to 8.4, and both hemes present a significant redox-Bohr effect. For instance, at pH 7, the midpoint reduction potentials of the hemes B and A₃ are 213 mV and 285 mV, respectively, whereas at pH 8.4, the order is reversed: 246 mV for heme B and 199 mV for heme A₃. The existence of redox anticooperativity was established by introducing a redox interaction parameter in a model of pairwise interacting redox centers.     

Substrate specificity,environmental degradation and disturbance structuring macroinvertebrate assemblages in neotropical streams

Structure and composition of benthic macroinvertebrate assemblages were investigated in seven sampling sites with a gradient of environmental integrity and water quality conditions. Composite samples of the four most representative substrates were collected in order to characterize the riffle-pool dynamic in each sampling site. Spatial and temporal variability of macroinvertebrate assemblages were analyzed at two scales: using substrates and grouping samples for comparing sampling sites. Distribution of macroinvertebrates was influenced primarily by substrate type, but also by environmental integrity, water quality and sampling period. Species occurrence was highly dependent on substrate type. At local spatial scale, environmental degradation measured by the Riparian Channel Environmental Inventory and water chemistry were the determinants of assemblage patterns. We evaluated to which extent the substrates were influenced by environmental integrity and water chemistry, and we found that degradation influenced significantly the macroinvertebrate fauna on the four substrate types, although they were not responding to the same variables. Our results show that qualitatively communities were not influenced by seasonal changes, but abundance was stochastically dependent on rainfall.     

Evidence of differential renal dysfunctions during exercise in men

Post-exercise proteinuria is a common phenomenon in healthy subjects. Previous studies have used albumin (Alb) and β₂-microglobulin (β₂-m) molecules as representatives of high- and low-molecular-weight proteins. Recently, more specific markers of the human kidney proximal tubule have been used to identify the precise site of alterations. Active male subjects underwent two strenuous runs, one 400-m run and one 3000-m run. Urine was collected from the subjects before and after each event. Total protein (TP), Alb, α₁-microglobulin (α₁-m), β₂-m, intestinal alkaline phosphatase (IAP), tissue-nonspecific alkaline phosphatase (TNAP) and N-acetyl-β-d-glucosaminidase (NAG) were determined for each sample. The short-distance run (400 m) resulted in the largest increases (P ≤ 0.05) in TP (31-fold), Alb (100-fold) and β₂-m (164-fold) as compared to the long-distance run (3000-m). The α₁-m excretion rates were increased to a lesser extent by the exercises. The IAP activity was slightly increased (+90%) by the 400-m run while the TNAP and NAG activities showed a 6.8-fold and a 3.6-fold increase, respectively, after this event. Smaller increases were recorded for the long-distance run (P = 0.05). To conclude, the present investigation showed that: (1) post-exercise proteinuria is related to the absolute intensity of exercise; (2) the impairment of protein reabsorption is revealed better by changes in Alb and β₂-m; (3) changes in TNAP and NAG activities could reveal biochemical modifications that occur in the proximal tubule, particularly at the S1-S2 segment. Accepted: 31 January 1997     

The role of the sexual organs in the generation of postembryonic neurons in the leech Hirudo medicinalis

In adult Hirudo medicinalis, the segmental ganglia (SG) of the fifth and sixth body segments contain a few hundred more cells than the other segmental ganglia of the body (Macagno, 1980). These ganglia innervate the sex organs and are known as the sex SG. As shown here, these cells are stained by neuron-specific antibodies. Cell counts at several developmental stages reveal that the extra cells are added exclusively to the sex SG, and that this addition occurs gradually and almost entirely postembryonically (embryogenesis ends at 30 days of development). Hence, we refer to them as PE cells. Until 16 days of development, deleting the male genitalia or disconnecting them from the sex ganglia results in a complete absence of the PE cells. From 16 days onward, the generation of the PE cells is independent of the male genitalia. No PE cells appear in non-sex SG that innervate male genitalia transplanted to ectopic locations at 10 days or later, indicating that as early as about 10 days of development sex SG are different from other SG in their ability to acquire PE cells. These data suggest that the PE cells are generated within the sex SG, in a process that is triggered by an interaction with the male genitalia and mediated through the innervation of the organs by these ganglia.     

Mutual Information Rate and Bounds for It

The amount of information exchanged per unit of time between two nodes in a dynamical network or between two data sets is a powerful concept for analysing complex systems. This quantity, known as the mutual information rate (MIR), is calculated from the mutual information, which is rigorously defined only for random systems. Moreover, the definition of mutual information is based on probabilities of significant events. This work offers a simple alternative way to calculate the MIR in dynamical (deterministic) networks or between two time series (not fully deterministic), and to calculate its upper and lower bounds without having to calculate probabilities, but rather in terms of well known and well defined quantities in dynamical systems. As possible applications of our bounds, we study the relationship between synchronisation and the exchange of information in a system of two coupled maps and in experimental networks of coupled oscillators.