In vitro conditions affect photosynthetic performance and crassulacean acid metabolism in Mammillaria gracilis Pfeiff. tissues

Mammillaria gracilis Pfeiff. plants cultivated in the pot (pot plants, PP), as well as in vitro-grown normal shoots (NS), habituated callus (HC), hyperhydric shoots (HS), and tumour tissue (TT) were investigated in order to reveal the influence of in vitro culture on functionality of the photosynthetic apparatus and CAM photosynthesis in cactus M. gracilis Pfeiff. Photosynthetic pigments content as well as maximum (F _v/F _m) and effective (??_PSII) quantum yield of photosystem II (PSII) decreased in all in vitro-grown tissues in comparison to PP. The decrease observed in hyperhydric HC, HS and TT correlated with a low expression of Rubisco large subunit (RbcL) and ?? subunit of ATP synthase (?? ATP synt) and almost undetectable levels of protein D1, light-harvesting chlorophyll a/b-binding protein (LHCII) and cytochrome f protein of thylakoid Cyt b ₆ /f-complex (Cyt f) found in these tissues. As for crassulacean acid metabolism (CAM) pattern, PP and NS expressed diurnal acid fluctuation, while HC, HS and TT failed to show it. Nevertheless, all M. gracilis tissues exhibited diurnal changes of phosphoenolpyruvate carboxylase (PEPC) activity indicating the typical CAM physiology. In conclusion, the photosynthesis was down-regulated in all in vitro-grown tissues. NS maintained typical CAM photosynthesis, while HC, HS and TT withheld PEPC activity, but not acid accumulation specific for CAM. Minor changes observed in NS in comparison to PP could be attributed to the sugar supplementation while the more prominent deviations found in HC, HS and TT could be correlated with hyperhydricity and the loss of characteristic tissue organisation pattern.     

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.     

A practical validation approach for virus titer testing of avian infectious bursal disease live vaccine according to current regulatory guidelines

The method for virus titer determination of avian infectious bursal disease (IBD) live vaccine, developed long before regulatory validation guidelines is a cell culture based biological assay intended for use in vaccine release testing.The aim of our study was to perform a validation, based on fit-for-purpose principle, of an old 50% tissue culture infectious dose (TCID₅₀) method according to Guidelines of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH).This paper addresses challenges and discusses some key aspects that should be considered when validating biological methods. A different statistical approach and non-parametric statistics was introduced in validation protocol in order to derive useful information from experimental data. This approach is applicable for a wide range of methods.In conclusion, the previous virus titration method had showed to be precise, accurate, linear, robust and in accordance with current regulatory standards, which indicates that there is no need for additional re-development or upgrades of the method for its suitability for intended use.     

Cadmium Uptake by Lettuce (Lactuca sativa L.) as Basis for Derivation of Risk Limits in Soils

The availability and uptake of Cd by lettuce (Lactuca sativa L.) in two common tropical soils (before and after liming) were studied in order to derive human health-based risk soil concentration. Cadmium concentrations ranging from 1 to 12 mg kg^?1 were added to samples from a clayey Oxisol and a sandy-loam Ultisol under glasshouse conditions. After incubation, a soil sample was taken from each pot, the concentration of Cd in the soil was determined, lettuce was grown during 36 d, and the edible parts were harvested and analyzed for Cd. A positive linear correlation was observed between total soil Cd and the Cd concentration in lettuce. The amount of Cd absorbed by lettuce grown in the Ultisol was about twice the amount absorbed in the Oxisol. Liming increased the soil pH and slightly reduced Cd availability and uptake. CaCl₂ extraction was better than DTPA to reflect differences in binding strength of Cd between limed and unlimed soils. Risk Cd concentrations in the Ultisol were lower than in the Oxisol, reflecting the greater degree of uptake from the Ultisol. The derived risk Cd values were dependent on soil type and the exposure scenario.     

Chitosan influence on glucose and calcium availability from yogurt: In vitro comparative study with plants fibre

Since chitosan complies with the definition of dietary fibre is necessary to study the interaction of this biopolymer with nutrients. Yogurt with fortified chitosan and different types of plants fibres like wheat, bamboo, apple, psyllium and inulin was used as a food model. The availabilities of glucose and calcium in this model were studied by an in vitro gastrointestinal tract simulation. Results showed that the different fibres decreased both glucose and calcium availabilities whereas the effect of chitosan was more pronounced. (17.7 ± 2.1% and 21.0 ± 2.5% depress respectively). This work demonstrated that the addition of chitosan to yogurts influences the availability of nutrients.     

Densitometric analysis of dental implant placement between flapless technique and the two-stage technique--a pilot study

Flapless technique is a surgical approach of implant placement without raising a mucoperiosteal flap. Such approach has many advantages: shorter surgical treatment, minimal bleeding, postoperative discomfort for the patient is reduced; possibility of immediate loading of the inserted implant, faster procedure of implant placement and by that less time is needed for the complete implant-prosthetic restoration. Purpose of this pilot study was radiographic assessment of flapless technique and determination of its clinical values in comparison with two-stage dental implant technique through computerized densitometric analysis. The sample consisted of 10 patients with missing teeth in the premolar region in the upper jaw. An implant was placed in that position. In the first group of 5 patients the implants were inserted with the flapless technique, and in the other group of 5 patients implant insertion was done with a two-stage technique. All inserted implants were loaded with metal-ceramic crowns 3 months after placement. The patients were followed for 18 months through clinical follow-ups and radiovisiographical (RVG) images made after 3, 12 and 18 months. After comparing the average densities, the results showed similar decrease of density in both groups, conventional two-stage technique showed 3.24 and flapless technique 1.23. It can be concluded that flapless technique in everyday clinical usage has the same result as the two-stage dental implant technique.     

Thermal stabilization of trypsin with glycol chitosan

Glycol chitosan was evaluated as thermoprotectant additive for trypsin in aqueous solutions. Maximal stabilization was achieved by using a polymer/protein ratio of 2 (w/w). The catalytic properties of trypsin were not affected by the presence of the polysaccharide. The enzyme thermostability was increased from 49 °C to 93 °C in the presence of the additive. Trypsin was also 37-fold more stable against incubation at 55 °C and its activation free energy of thermal inactivation was increased by 9.9 kJ/mol when adding glycol chitosan.     

Modeling changes in soil organic matter in Amazon forest to pasture conversion with the Century model

Land use and land cover changes in the Brazilian Amazon region have major implications for regional and even global carbon cycling. We analyzed the effects of the predominant land use change, conversion of tropical forest to pasture, on total soil C and N, using the Century ecosystem model and data collected from the Nova Vida ranch, Western Brazilian Amazon. We estimated equilibrium organic matter levels, plant productivity and residue carbon inputs under native forest conditions, then simulated deforestation following the slash and burn procedure. Soil organic matter dynamics were simulated for pastures established in 1989, 1987, 1983, 1979, 1972, 1951, and 1911. Using input data from the Nova Vida ranch, the Century model predicted that forest clearance and conversion to pasture would cause an initial decline in soil C and N stocks, followed by a slow rise to levels exceeding those under native forest. Simulated soil total C and N levels (2500 g C m^?2 and 245 g N m^?2 in the 0–20 cm layer) prior to conversion to pasture were close to those measured in the native forest. Simulated above‐ and below‐ground biomass for the forest and pasture were comparable with literature values from this region. The model predicted the long‐term changes in soil C and N under pasture inferred from the pasture chronosequence, but there was considerable variation in soil C stocks for pastures <20 years in age. Differences in soil texture between pastures were relatively small and could not account for much of the variability between different pastures of similar ages, in either the measured or simulated data. It is likely that much of the variability in C stocks between pastures of similar ages is related to initial C stocks immediately following deforestation and that this was the largest source of variability in the chronosequence. Internal C cycling processes in Century were evaluated using measurements of microbial biomass and soil δ¹³C. The relative magnitude and long‐term trend in microbial biomass simulated by the model were consistent with measurements. The close fit of simulated to measured values of δ¹³C over time suggests that the relative loss of forest‐derived C and its replacement by pasture‐derived C was accurately predicted by the model. After 80 years, almost 90% of the organic matter in the top 20 cm was pasture derived. While our analysis represents a single ‘case study’ of pasture conversion, our results suggest that modeling studies in these pasture systems can help to evaluate the magnitude of impacts on C and N cycling, and determine the effect of management strategies on pasture sustainability.     

GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection

Multiple sclerosis (MS) is a highly disabling neurodegenerative autoimmune condition in which an unbalanced immune response plays a critical role. Although the mechanisms remain poorly defined, helminth infections are known to modulate the severity and progression of chronic inflammatory diseases. The tyrosine kinase receptors TYRO3, AXL, and MERTK (TAM) have been described as inhibitors of the immune response in various inflammatory settings. We show here that patients with concurrent natural helminth infections and MS condition (HIMS) had an increased expression of the negative regulatory TAM receptors in antigen-presenting cells and their agonist GAS6 in circulating CD11b^high and CD4⁺ T cells compared to patients with only MS. The Th17 subset was reduced in patients with HIMS with a subsequent downregulation of its pathogenic genetic program. Moreover, these CD4⁺ T cells promoted lower levels of the co-stimulatory molecules CD80, CD86, and CD40 on dendritic cells compared with CD4⁺ T cells from patients with MS, an effect that was GAS6-dependent. IL-10⁺ cells from patients with HIMS showed higher GAS6 expression levels than Th17 cells, and inhibition of phosphatidylserine/GAS6 binding led to an expansion of Th17 effector genes. The addition of GAS6 on activated CD4⁺ T cells from patients with MS restrains the Th17 gene expression signature. This cohort of patients with HIMS unravels a promising regulatory mechanism to dampen the Th17 inflammatory response in autoimmunity.     

Selection of reference genes for reverse transcription-qPCR analysis in the biomonitor macrophyte <Emphasis Type="Italic">Bidens laevis</Emphasis> L.

The RT-qPCR has been the method used to analyze gene expression in plants but its benefits have not been completely exploited in the field of plants ecotoxicology when used as molecular biomarkers. The correct use of RT-qPCR demands to establish a certain number of reference genes (RG) which are expected to be invariable in their expression although it does not always happen. The main goals of this work were to: (1) analyze the stability of six potential RG, (2) establish the optimum number of RG, (3) select the most suitable RG to be applied in Bidens laevis under different test conditions and tissues and (4) confirm its convenience by normalizing the expression of one gene of interest under three different challenges. When all data were pooled together, the geNorm algorithm pointed out beta-actin and beta-tubulin (TUB) as the optimal RG pair while NormFinder algorithm selected nicotinamide adenine dinucleotide dehydrogenase (NADHD) and histone 3 (H3) as possessing the most invariable levels of expression. On the other hand, when data were grouped by tissues, ANOVA test selected H3 and TUB, while data grouped by conditions indicated that H3 and NADHD were the most stable RG under this analysis. Therefore, for a general-purpose set of RG, the overall analysis showed that a set of three RG would be optimum, and H3, TUB and NADHD were the selected ones. On the other hand, as RG can vary depending on the tissues or conditions, results achieved with ANOVA would be more reliable. Thus, appropriate normalization process would clearly need more than one RG.