Characteristics of in vivo murine erythropoietic response to sodium orthovanadate

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.     

Calsequestrin (CASQ1) rescues function and structure of calcium release units in skeletal muscles of CASQ1-null mice

Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.     

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.     

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.     

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.     

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.     

Experimental analysis of the effects of consumer exclusion on recruitment and succession of a coral reef system along a water quality gradient in the Spermonde Archipelago,Indonesia

Coral Reefs - The composition of coral reef benthic communities is strongly affected by variation in water quality and consumer abundance and composition. This is particularly evident in highly...