Deciphering the Prokaryotic Community and Metabolisms in South African Deep-Mine Biofilms through Antibody Microarrays and Graph Theory

In the South African deep mines, a variety of biofilms growing in mine corridor walls as water seeps from intersections or from fractures represents excellent proxies for deep-subsurface environments. However, they may be greatly affected by the oxygen inputs through the galleries of mining activities. As a consequence, the interaction between the anaerobic water coming out from the walls with the oxygen inputs creates new conditions that support rich microbial communities. The inherent difficulties for sampling these delicate habitats, together with transport and storage conditions may alter the community features and composition. Therefore, the development of in situ monitoring methods would be desirable for quick evaluation of the microbial community. In this work, we report the usefulness of an antibody-microarray (EMChip66) immunoassay for a quick check of the microbial diversity of biofilms located at 1.3 km below surface within the Beatrix deep gold mine (South Africa). In addition, a deconvolution method, previously described and used for environmental monitoring, based on graph theory and applied on antibody cross-reactivity was used to interpret the immunoassay results. The results were corroborated and further expanded by 16S rRNA gene sequencing analysis. Both culture-independent techniques coincided in detecting features related to aerobic sulfur-oxidizers, aerobic chemoorganotrophic Alphaproteobacteria and metanotrophic Gammaproteobacteria. 16S rRNA gene sequencing detected phylotypes related to nitrate-reducers and anaerobic sulfur-oxidizers, whereas the EMChip66 detected immunological features from methanogens and sulfate-reducers. The results reveal a diverse microbial community with syntrophic metabolisms both anaerobic (fermentation, methanogenesis, sulphate and nitrate reduction) and aerobic (methanotrophy, sulphur oxidation). The presence of oxygen-scavenging microbes might indicate that the system is modified by the artificial oxygen inputs from the mine galleries.     

A modified staining technique for arbuscular mycorrhiza compatible with molecular probes

The effects of the different steps of the root staining on the arbuscular mycorrhizal (AM) fungal rDNA extraction and amplification have been assessed. The results obtained using molecular techniques are compared with those obtained from fresh, non-stained leek roots. A modified staining procedure that eliminates heating, the use of hydrochloric acid and trypan blue, has been proved to be the most adequate to observe the AM colonisation in different plant species with/without lignified roots allowing at the same time the subsequent rDNA extraction and amplification from the stained roots. The staining technique decreased the sensitivity of the process and a higher number of roots had to be used to obtain enough material for a positive amplification. The extraction and amplification process was reliable up to 3 days after staining. A week after staining, the amplification was not dependable and after 2 weeks there was no amplification from stained material.     

A novel chromate reductase from Thermus scotoductus SA-01 related to old yellow enzyme

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.     

Reactivity of isolated human chorionic vessels: analysis of some influencing variables

The aim of the study was to determine whether 24 h of cold storage of samples, mode of delivery, and gestational age influenced in vitro human chorionic vascular reactivity (35 arteries and 34 veins). The following groups were compared: (i) fresh versus 24-h cold-stored (4 degrees C in Krebs-Henseleit solution) chorionic vascular rings from normal term placentas, (ii) fresh chorionic vascular rings from normal term placentas obtained after vaginal delivery versus those obtained after elective caesarean section, and (iii) fresh chorionic vascular rings from normal term placentas versus those obtained from preterm deliveries. Isometric recording of the concentration-response curve to KCl (5-120 mM) was assessed in each group. In vitro human chorionic vascular reactivity was influenced negatively by the 24-h cold storage of samples, with only 30% of stored samples being weakly reactive to KCl. Human chorionic vascular reactivity to KCl was unaffected by the mode of delivery. However, the response to KCl was gestational-age dependent. Thus, preterm vascular rings exhibited a significantly (P<0.05) decreased response (Emax=9.8 +/- 0.0 mN; EC50=26.0 +/- 1.3 mM) compared with term samples (Emax=21.6 +/- 2 mN; EC50=13.9 +/- 1.6 mM). In conclusion, this study provides evidence that fresh term vascular rings are the tissues of choice for studying human chorionic vascular reactivity.     

Effects of dopamine in isolated rat colon strips

The aim of the present work is to investigate the effects of dopamine on isolated rat colon strips, and whether dopamine receptors are involved in these effects. Experiments on spontaneous motility and under potassium contraction were performed with dopamine and isoprenaline, both in the absence and presence of antagonists (distal colon strips, isotonic recording, Tyrode solution, 31 degrees C, 1 g of resting tension). At higher concentration (10(-4) mol/L), dopamine abolished spontaneous motility of the rat colon and this effect was not modified by antagonists. In isolated rat colon strips that were depolarized with potassium, dopamine produced concentration-dependent relaxation, without significant differences in reserpinized rats. Preincubation with sulpiride or Sch 23390, dopamine antagonists, did not modify the effects of dopamine. Propranolol shifted the concentration-response curve to the right, though in a noncompetitive manner. Prazosin and yohimbine (alpha-antagonists) did not modify the response to dopamine. Isoprenaline produced a concentration-dependent relaxant response to the KCl-induced contraction antagonized by propranolol, but not by prazosin, in a noncompetitive manner. In conclusion, dopamine exhibits a relaxant effect on the isolated rat colon, which is not mediated by specific dopamine receptors or alpha-adrenoceptors but it may be mediated by atypical beta-adrenoceptors.     

Response of tomato cultivars to salinity

The responses of five tomato cultivars (L. esculentum Mill) of different degrees of salt tolerance were examined over a range of 0 to 140 mM NaCl applied for 3 and 10 weeks. Judged by both Na and Cl accumulations and maintenance of K, Ca and Mg contents with increasing salinity, the most tolerant cultivars (Pera and GC-72) showed different responses. The greater salt tolerance of cv Pera was associated with a higher Cl and Na accumulation and a lower K content in the shoot than those found in the other cultivars, typical of a halophytic response to salinity. However, the greater salt tolerance of cv GC-72 was associated with a retention of Na and Cl in the root, restriction of their translocation to the shoot and maintenance of potassium selectivity under saline conditions. The salt tolerance mechanisms that operated in the remaining cultivars were similar to that of cv GC-72, as at first they excluded Na and Cl from the shoots, accumulating them in the roots; with longer treatment, the ability to regulate Na and Cl concentrations in the plant was lost only in the most salt sensitive cultivar (Volgogradskij), resulting in a massive influx of both ions into the shoot.The salt sensitivity of some tomato cultivars to salinity could be due to both the toxic effect of Na and Cl ions and nutritional imbalance induced by salinity, as plant growth was inversely correlated with Na and Cl contents and directly correlated with K and Ca contents. This study displays that there is not a single salt tolerance mechanism, since different physiological responses among tomato cultivars have been found.     

Salinity tolerance of normal-fruited and cherry tomato cultivars

The salinity tolerances (NaCl) of 8 normal-fruited tomato cultivars (Lycopersicon esculentum Mill.) and 4 cherry tomato cultivars (L. esculentum var.cerasiforme) were determined by yield-substrate EC response curves, according to the Mass-Hoffman model, modified by van Genuchten and Hoffman (1984). The same model was used to determine the response curves of leaf dry-weight, stem dry-weight, and plant height against substrate EC and also between yield and leaf concentrations of Cl- and Na ions.According to the salinity-threshold (maximum EC-value without yield reduction) and slope (yield decrease per unit EC increase) parameters, determined from the yield-EC response curves, the cherry tomato cultivars were more salt-tolerant than the normal-fruited ones. However, on the basis of vegetative growth characters-EC response curves, cherry tomato cultivars and normal-fruited ones were similarly affected by NaCl.The ranking of the cultivars by their salinity tolerance, determined from the plots of yield vs. leaf concentrations of Cl- and Na ions, was the same as that evaluated from the yield vs. substrate EC plots.     

Realistic Ensemble Models of Intrinsically Disordered Proteins Using a Structure-Encoding Coil Database

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NaCl pre-treatment at the seedling stage enhances fruit yield of tomato plants irrigated with salt water

Although salt-adaptation seems to be a widespread property of plants, the adaptive response has been rarely differentiated to the tolerance response. We report on the adaptive response of tomato plants to growing under saline conditions following a 15 day pre-treatment with a lower NaCl concentration (half) than that used during the plant growth. After 20 days of salt treatment (100 mM NaCl), the biomass of the adapted plants increased significantly with respect to that of the unadapted plants when the pre-treatment was applied to five leaf seedlings, but not at the two leaf stage. The long-term adaptive response was determined in two tomato genotypes with different tolerance to moderate salt levels. At 70 mM NaCl, the adapted-plants of the more salt-sensitive genotype produced up to 29% more fruit yield than did the unadapted plants. However, no positive effect was observed to long-term in the adapted-plants of the more salt-tolerant genotype, which suggests that the stress level necessary to trigger the adaptive response is related to the tolerance degree of genotype. The physiological response of the plants showing a positive response to the adaptation was also modified to long-term. Thus, K⁺ concentrations increased in the young leaves of the adapted plants, with respect to unadapted plants, and moreover these differences increased with the salinization period. These results indicate that the changes in growth and physiological responses induced by NaCl pre-treatment at the seedling stage are maintained throughout plant life cycle and this is, therefore, an interesting strategy for increasing the salt tolerance in tomato plants.