Constraints in the identification and interpretation of ozone as a significant predictor of effects on the supersensitive indicator Nicotiana tabacum Bel-W3 in biomonitoring studies

Biomonitoring by means of the supersensitive cultivar Nicotiana tabacum Bel-W3 is frequently used to obtain information on ozone effects on plants and estimates of ozone exposure. However, most of biomonitoring surveys do not account for other environmental variables (predictors in a statistical model) and their inherent multicollinearity with ozone. We tested the relative role of different predictors (fixed: time and site; random: ozone, temperature and humidity) on height growth and on the development of visible foliar symptoms of N. tabacum Bel-W3 plants. To do this, we investigated a relatively small area (256 km²), used a random design at every stage of the survey, controlled watering and protected plants from direct solar radiation and wind. QA/QC procedures were adopted at every stage of the investigation. Linear correlation shows that Leaf Injury Index (LII) and height increment (H.I.) positively related to ozone concentration, elevation and temperature, and negatively to relative humidity. All the predictors correlate to each other. However, relationships between response and ozone vary with the site and the monitoring week. The effect of the random factor “ozone” in combination with fixed factors “site” and “time” on the response variables was therefore formally investigated using the ANCOVA model. Besides ozone, the interactions “ozone × site” and “ozone × time” resulted always significant (0.001 < P < 0.05). While the factor “time” emphasize the inherent development of injury and growth through time, the interaction “ozone × site” pointed out the importance of local conditions. When watering, solar radiation, wind and plant characteristics were controlled, the remaining site-specific covariates of interest were temperature (T) and humidity (RH). When T and RH were accounted for by means of partial correlation analysis, no significant relationship was found between ozone and LII. On the other side, O₃ and RH resulted significant for both absolute and relative height increment. In short, LII seemed to be not solely dependent on ozone, T and RH, but showed to integrate their combined effect. On the other side, H.I. seemed to be favoured by high RH and T, and depressed by high ozone. Based on the above results, we recommend caution when handling bioindicator data: if the purpose is to infer ozone concentrations by leaf injury data, results may be affected by a serious bias, as the frequently reported correlations may be partly an artefact due to co-variation between predictors.     

High mitochondrial mutation rates estimated from deep-rooting Costa Rican pedigrees

Estimates of mutation rates for the noncoding hypervariable Region I (HVR-I) of mitochondrial DNA vary widely, depending on whether they are inferred from phylogenies (assuming that molecular evolution is clock-like) or directly from pedigrees. All pedigree-based studies so far were conducted on populations of European origin. In this article, we analyzed 19 deep-rooting pedigrees in a population of mixed origin in Costa Rica. We calculated two estimates of the HVR-I mutation rate, one considering all apparent mutations, and one disregarding changes at sites known to be mutational hot spots and eliminating genealogy branches which might be suspected to include errors, or unrecognized adoptions along the female lines. At the end of this procedure, we still observed a mutation rate equal to 1.24 × 10(-6) , per site per year, i.e., at least threefold as high as estimates derived from phylogenies. Our results confirm that mutation rates observed in pedigrees are much higher than estimated assuming a neutral model of long-term HVRI evolution. We argue that until the cause of these discrepancies will be fully understood, both lower estimates (i.e., those derived from phylogenetic comparisons) and higher, direct estimates such as those obtained in this study, should be considered when modeling evolutionary and demographic processes.     

Photosynthetic changes and oxidative stress caused by iron ore dust deposition in the tropical CAM tree Clusia hilariana

The effect of iron solid particulate matter (SPM_Fe) deposited onto soil and leaves on photosynthesis and oxidative stress was evaluated in Clusia hilariana, a CAM tropical tree of high occurrence in Brazilian restingas. Significant increases in iron content were found in plants exposed to SPM_Fe applied onto leaf and soil surfaces. However, only the application of SPM_Fe on leaves of C. hilariana caused significant reductions in some evaluated characteristics such as photosynthetic rate, stomatal conductance, transpiration, organic acid accumulation, potential quantum yield of PSII, and changes in daily CAM photosynthesis pattern. Increase in relative membrane permeability and reduction in catalase and superoxide dismutase activities in the leaves of plants exposed to SPM_Fe also were observed; however, lipid peroxidation did not change. These responses seem to be due to the combination of physical effects such as increase of leaf temperature, reduction in light absorption, obstruction of stomatal pores, and biochemical effects triggered by oxidative stress.     

Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.     

Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found: Lactobacillus fermentum strains F53 and KC5b, Bifidobacterium infantis ATCC 15697, Streptococcus bovis ATCC 43143, Enterococcus durans DSM 20633, Enterococcus gallinarum, and Enterococcus faecalis. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate L. fermentum KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.     

A reliable methodology for assessing the in vitro photosynthetic competence of two Brazilian savanna species: Hyptis marrubioides and Hancornia speciosa

Evaluation of photosynthetic efficiency is critical for studies on plant responses to environmental conditions as well as for genotype selection; however, there is a lack of reliable and functional protocols for such assessments of plants cultured in vitro. In this study, we aimed to adapt the conventional methodology for measuring gas exchange of plants grown in vitro to analyze the effects of irradiance, flow rate, and air humidity on the photosynthetic rate in cultured plantlets of two ‘Cerrado’ species, namely Hyptis marrubioides and Hancornia speciosa plantlets. Chlorophyll (chl) a fluorescence and chloroplastidic pigment content were also assessed. The highest photosynthetic rates were observed at a photon flux density of 600 μmol m^?2 s^?1, with tube inlet airflow rates between 100 and 300 mL min^?1 and 80 % relative humidity in the inlet air. The electron transport rate curve, by means of chl a fluorescence, was similar to the photosynthetic rate response curve obtained with the infrared gas analyzer. These results demonstrate that both H. marrubioides and H. speciosa seedlings grown in vitro have a functional photosynthetic apparatus and respond to variations in measurement conditions, exhibiting substantial rates of CO₂ assimilation under saturating irradiance conditions. The methodology proposed here can be adapted and applied to other species growing in vitro.     

Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor

Parameters of VERO cell growth and metabolism were studied in cultures performed on microcarriers (MCs) using a bioreactor with a working capacity of 3.7?l. Kinetic studies of VERO cell growth in batch, semi-batch and perfusion cultures using concentrations of 2 and 10?mg/ml of MCs showed that a high concentration of MCs (10?mg/ml) and the use of medium perfusion allowed the attainment of higher final yields of VERO cells (6?×?10⁶ cells/ml after 10 days of culture). Perfusion also allowed better use of MCs as indicated by the observation of about 100% of MCs totally covered by cells and the appearance of multilayered cells on 64% of MCs after 13 days of VERO cell culture with 2?mg/ml of MCs. Concerning the concentration of nutrients in the cultures, the medium perfusion was able to sustain suitable levels of galactose and glutamine, which quickly decreased after 4 days in batch cultures. The air inlet in the batch cultures was capable of eliminating the NH₄ ⁺ which accumulated in the medium culture. Lactate accumulated during the first days of culture but then was utilized by the cells and decreased along the culture time. The optimization of VERO cell cultures on microcarriers as indicated by the concentration of MCs, medium perfusion and air inlet is discussed.