In vitro evaluation, in vivo quantification, and microbial diversity studies of nutritional strategies for reducing enteric methane production

The main objective of the present work was to study nutritive strategies for lessening the CH4 formation associated to ruminant tropical diets. In vitro gas production technique was used for evaluating the effect of tannin-rich plants, essential oils, and biodiesel co-products on CH4 formation in three individual studies and a small chamber system to measure CH4 released by sheep for in vivo studies was developed. Microbial rumen population diversity from in vitro assays was studied using qPCR. In vitro studies with tanniniferous plants, herbal plant essential oils derived from thyme, fennel, ginger, black seed, and Eucalyptus oil (EuO) added to the basal diet and cakes of oleaginous plants (cotton, palm, castor plant, turnip, and lupine), which were included in the basal diet to replace soybean meal, presented significant differences regarding fermentation gas production and CH4 formation. In vivo assays were performed according to the results of the in vitro assays. , when supplemented to a basal diet (Tifton-85 hay sp, corn grain, soybean meal, cotton seed meal, and mineral mixture) fed to adult Santa Ines sheep reduced enteric CH4 emission but the supplementation of the basal diet with EuO did not affect ( > 0.05) methane released. Regarding the microbial studies of rumen population diversity using qPCR with DNA samples collected from the in vitro trials, the results showed shifts in microbial communities of the tannin-rich plants in relation to control plant. This research demonstrated that tannin-rich , essential oil from eucalyptus, and biodiesel co-products either in vitro or in vivo assays showed potential to mitigate CH4 emission in ruminants. The microbial community study suggested that the reduction in CH4 production may be attributed to a decrease in fermentable substrate rather than to a direct effect on methanogenesis.

MR spectroscopy in human prostate: in vitro and in vivo measurements to optimize new quantification algorithms

Il cancro della prostata (PCa) è il tumore maligno non-cutaneo più diffuso tra gli uomini ed è il secondo tumore che miete più vittime nei paesi occidentali. La necessità di nuove tecniche non invasive per la diagnosi precoce del PCa è aumentata negli anni. 1H-MRS (proton magnetic resonance spectroscopy) e 1H-MRSI (proton magnetic resonance spectroscopy imaging) sono tecniche avanzate di spettroscopia in risonanza magnetica che permettono di individuare presenza di metaboliti come citrato, colina, creatina e in alcuni casi poliammine in uno o più voxel nel tessuto prostatico. L’abbondanza o l’assenza di uno di questi metaboliti rende possibile discriminare un tessuto sano da uno patologico. Le tecniche di spettroscopia RM sono correntemente utilizzate nella pratica clinica per cervello e fegato, con l’utilizzo di software dedicati per l’analisi degli spettri. La quantificazione di metaboliti nella prostata invece può risultare difficile a causa del basso rapporto segnale/rumore (SNR) degli spettri e del forte accoppiamento-j del citrato. Lo scopo principale di questo lavoro è di proporre un software prototipo per la quantificazione automatica di citrato, colina e creatina nella prostata. Lo sviluppo del programma e dei suoi algoritmi è stato portato avanti all’interno dell’IRST (Istituto Romagnolo per lo Studio e la cura dei Tumori) con l’aiuto dell’unità di fisica sanitaria. Il cuore del programma è un algoritmo iterativo per il fit degli spettri che fa uso di simulazioni MRS sviluppate con il pacchetto di librerie GAMMA in C++. L’accuratezza delle quantificazioni è stata testata con dei fantocci realizzati all’interno dei laboratori dell’istituto. Tutte le misure spettroscopiche sono state eseguite con il nuovo scanner Philips Ingenia 3T, una delle machine di risonanza magnetica più avanzate per applicazioni cliniche. Infine, dopo aver eseguito i test in vitro sui fantocci, sono stati acquisiti gli spettri delle prostate di alcuni volontari sani, per testare se il programma fosse in grado di lavorare in condizioni di basso SNR.

Quantification of Mitral Regurgitation with Multiple Jets: In Vitro Comparison of Two-dimensional PISA techniques

Purpose: Traditionally, the proximal isovelocity surface area (PISA) is based on the assumption of a single hemisphere (hemispheric PISA), but this technique has not been validated for the quantification of mitral regurgitation (MR) with multiple jets. Methods: The left heart simulator was actuated by a pulsatile pump at various stroke amplitudes. The regurgitant volume (Rvol) passing through the mitral valve phantoms with single and double regurgitant orifices of varying size and interspace was quantified by a flowmeter as reference technique. Color Doppler 3-D full-volumes were obtained, and Rvol were derived from 2-D PISA surfaces on the basis of hemispheric and hemicylindric assumption with one base (partial hemicylindric PISA) or 2 bases (total hemicylindric PISA). Results: 72 regurgitant volumes (Rvol range: 8 to 76 ml/beat) were obtained. Hemispheric PISA Rvol correlated well with reference Rvol by one orifice (R²=0.97; bias -2.7±3.2ml), but less by ≥ one orifice (R²=0.89). When a fusion of two PISAs occured, addition of two hemispheric PISA overestimated Rvol (bias 9.1±12.2ml, fig.1), and single hemispheric PISA underestimated Rvol (bias -12.4±4.9ml). If an integrated approach was used (hemispheric in single orifice, total hemicylindric in two non-fused PISAs and partial hemicylindric in two fused PISAs), the correlation was R²=0.95, bias -1.6±5.6ml (fig.2). In the ROC analysis, the cutoff to detect ≥ moderate-to-severe Rvol (≥45ml) was 42ml (AUC 0.99, sens. 100%, spec. 93%). Conclusions: In MR with two regurgitant jets, the 2-D hemicylindric assumption of the PISA offers a better quantification of Rvol than the hemispheric assumption. Quantification of MR using 2-D PISA requires an integrated approach that considers number of regurgitant orifices and fusion of the PISAs.

Growth factor-loaded microparticles for tissue engineering: The discrepancies of in vitro characterization assays

Efficient and effective growth factor (GF) delivery is an ongoing challenge for tissue regeneration therapies. The accurate quantification of complex molecules such as GFs, encapsulated in polymeric delivery devices, is equally critical and just as complex as achieving efficient delivery of active GFs. In this study, GFs relevant to bone tissue formation, vascular endothelial growth factor (VEGF) and bone morphogenetic protein 7 (BMP-7), were encapsulated, using the technique of electrospraying, into poly(lactic-co-glycolic acid) microparticles that contained poly(ethylene glycol) and trehalose to assist GF bioactivity. Typical quantification procedures, such as extraction and release assays using saline buffer, generated a significant degree of GF interactions, which impaired accurate assessment by enzyme-linked immunosorbent assay (ELISA). When both dry BMP-7 and VEGF were processed with chloroform, as is the case during the electrospraying process, reduced concentrations of the GFs were detected by ELISA; however, the biological effect on myoblast cells (C2C12) or endothelial cells (HUVECs) was unaffected. When electrosprayed particles containing BMP-7 were cultured with preosteoblasts (MC3T3-E1), significant cell differentiation into osteoblasts was observed up to 3 weeks in culture, as assessed by measuring alkaline phosphatase. In conclusion, this study showed how electrosprayed microparticles ensured efficient delivery of fully active GFs relevant to bone tissue engineering. Critically, it also highlights major discrepancies in quantifying GFs in polymeric microparticle systems when comparing ELISA with cell-based assays.

Formação in vitro de biofilme de Candida albicans em materiais usados no preenchimento dos acessos aos parafusos das próteses sobre implantes

Os orifícios de acesso aos parafusos de retenção devem ser preenchidos para que o parafuso não seja danificado caso seja necessária a remoção da prótese. Dentre os materiais mais utilizados estão o algodão, a fita de politetrafluoretileno e a guta percha. O objetivo deste estudo é avaliar a formação de biofilme de Candida albicans nos materiais anteriormente descritos, buscando estabelecer um parâmetro que contribua para a escolha do tipo de material mais adequado a ser utilizado clinicamente. Foram utilizados UCLAs, análogos e parafusos sextavados, todos de titânio. Os conjuntos foram montados com torque de 32N. Os materiais foram condensados no interior dos UCLAs e colocados em meio de cultura com uma suspensão de 3x106 células/ml de Candida albicans. O sistema foi armazenado à 37C com agitação, por 15 dias e o meio foi renovado a cada 48 horas. A quantificação de biofilme foi realizada pelo ensaio de MTT e leitura à 490nm, resultando em diferentes valores de densidade óptica. A normalidade (p=0,304 - Kolmogorov-Smirnov) e a igualdade de variâncias (p=0,721 - Scheffe) foram testadas primeiramente. O teste de análise de variância demonstrou diferença significativa entre os grupos (p<0,001) e com o Holm-Sidak foi observada diferença significativa entre os grupos algodão e guta (p<0,05) e algodão e fita de politetrafluoetileno (p<0,05); não houve diferença significativa entre os grupos guta e fita de politatrafluoretileno (p>0,05), apesar dos valores da fita de politetrafluoetileno terem sido maiores. Considerando-se as limitações deste estudo in vitro, podemos concluir que tanto a guta-percha quanto a fita de politetrafluoretileno apresentaram menor formação de biofilme, não havendo diferença estatisticamente significativa entre os materiais. O algodão apresentou um nível de formação de biofilme significativamente maior que a fita de politetrafluoretileno e a guta percha. Diante disso, serão necessários novos estudos para confirmar as limitações que este tipo de material pode apresentar quando usado como material de preenchimento do acesso do parafuso da prótese sobre implante.

EFFECTS OF ENVIRONMENTAL ANTIOXIDANTS ON PRO-ANGIOGENIC ROS SIGNALLING IN ENDOTHELIAL COLONY FORMING CELLS IN VITRO

Introduction. Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Whilst increasing evidence supports a key role for reactive oxygen species (ROS), specifically those derived from Nox NADPH oxidases, in the underlying angiogenic processes of these and other endothelial cells, such studies investigating the role of redox signalling may be hampered by the standard inclusion of antioxidant agents in endothelial cell media, such as phenol red. Aims. To study the effects of antioxidants present in culture media on pro-angiogenic function of ECFCs in vitro. Methods. Human ECFCs isolated from umbilical cord blood were maintained in media with and without antioxidant components (EGM2 and phenol red-free DMEM, respectively) prior to treatment with pro-oxidant PMA and assessment of their in vitro migratory capacity using a scratch-wound assay to measure pro-angiogenic activity. Results. Our previous work in our group indicated that PMA (500nM) increased ECFC migration in a both a superoxide and NADPH oxidase-dependent manner (control 18.6±2.8, PMA 32.7±6.6% wound closure; n=6, P<0.05), as indicated by attenuation with PEG-SOD and VAS2870. However, inconsistencies in the data generated under varying experimental conditions led us to hypothesise that antioxidant agents in the standard ECFC media may be influencing these effects. Indeed, a direct comparison of cell migration between ECFCs incubated in EGM2 DMEM demonstrated a clear trend towards higher migration in the latter (EGM2 9.0±4.5, DMEM 22.7±6.4%; n=3, P=NS). Similar to our previous EGM2 studies, cell migration was potentiated by PMA (control 11.6±1.6, PMA 25.1±2.8%; n=3, P<0.05), but at a lower dose (100nM), which is consistent with a reduction in media antioxidants. Notably, this response was attenuated by VAS2870 (PMA 37.6±7.3, PMA+VAS2870 10.3±2.9%; n=6, P<0.05), underlining a likely role for Nox NADPH oxidases. Conclusion. Taken together, these data indicate that ECFC migration is sensitive to different endothelial cell growth media, which appears to be dependent upon their antioxidant content. Although further experiments, such as quantification of cellular superoxide generation by dihydroethidium fluorescence may be required to confirm a specific role for antioxidants, such blunting of ROS signalling in vitro is clearly an important consideration which may significantly impact upon data interpretation.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological. criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

In vitro evaluation of Cu and Fe bioavailability in cashew nuts by off-line coupled SEC-UV and SIMAAS

In this work Cu and Fe bioavailability in cashew nuts was evaluated using in vitro method. Extractions with simulated gastric and intestinal fluids and dialysis procedures were applied for this purpose. The proteins separation and quantification were performed by size exclusion chromatography (SEC) coupled on-line to ultra-violet (UV) and off-line to simultaneous multielement atomic absorption spectrometry (SIMAAS). The SEC-UV and SIMAAS profiles of the protein fractions obtained by alkaline extraction (NaOH) and precipitation with HCl indicated the presence of high and low molecular weight species in the range between >75 kDa and 9.3 kDa. Almost 83% of Cu and 78% of Fe were extracted during cashew nut digestion and 90% of both elements were dialyzed. With these results it is possible to assume that 75% of Cu and 70% of Fe present in cashew nut could be bioavailable. The SEC-UV and SIMAAS chromatographic profiles obtained after in vitro gastrointestinal digestion reveal that Cu and Fe not dialyzed can be associated to a compound of 9.2 kDa. (C) 2010 Elsevier B.V. All rights reserved.

Morbidity-mortality and performance evaluation of Brahman calves from in vitro embryo production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133(+) stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10 (13) mol iron (9.5 pg) or 7.0 x 10 (6) nanoparticles per cell ( the measurement was carried out in a volume of 2 mu L containing about 6.16 x 10 5 pg iron, equivalent to 4.5 x 10 (11) SPIONs). (c) 2008 Elsevier B.V. All rights reserved.

In vitro evaluation of the action of irrigating solutions associated with intracanal medications on Escherichia coli and its endotoxin in root canals

Objective: The purpose of this study was to evaluate the efficacy of auxiliary chemical substances and intracanal medications on Escherichia coli and its endotoxin in root canals. Material and Methods: Teeth were contaminated with a suspension of E. coli for 14 days and divided into 3 groups according to the auxiliary chemical substance used: G1) 2.5% sodium hypochlorite (NaOCl); G2) 2% chlorhexidine gel (CLX); G3) pyrogen-free solution. After, these groups were subdivided according to the intracanal medication (ICM): A) Calcium hydroxide paste (Calen (R)), B) polymyxin B, and C) Calcium hydroxide paste+2% CLX gel. For the control group (G4), pyrogen-free saline solution was used without application of intracanal medication. Samples of the root canal content were collected immediately after biomechanical preparation (BMP), at 7 days after BMP, after 14 days of intracanal medication activity, and 7 days after removal of intracanal medication. The following aspects were evaluated for all collections: a) antimicrobial activity; b) quantification of endotoxin by the limulus Amebocyte lysate test (LAL). Results were analyzed by the kruskal-wallis and Dunn's tests at 5% significance level. Results: The 2.5% NaOCl and CLX were able to eliminate E. coli from root canal lumen and reduced the amount of endotoxin compared to saline. Conclusions: It was concluded that 2.5% NaOCl and CLX were effective in eliminating E. coli. Only the studied intracanal medications were to reduce the amount of endotoxin present in the root canals, regardless of the irrigant used.

Photodynamic inactivation of Staphylococcus aureus and Escherichia coli biofilms by malachite green and phenothiazine dyes: An in vitro study

Objectives: The organization of biofilms in the oral cavity gives them added resistance to antimicrobial agents. The action of phenothiazinic photosensitizers on oral biofilms has already been reported. However, the action of the malachite green photosensitizer upon biofilm-organized microorganisms has not been described. The objective of the present work was to compare the action of malachite green with the phenothiazinic photosensitizers (methylene blue and toluidine blue) on Staphylococcus aureus and Escherichia coli biofilms.Methods: The biofilms were grown on sample pieces of acrylic resin and subjected to photodynamic therapy using a 660-nm diode laser and photosensitizer concentrations ranging from 37.5 to 3000 mu M. After photodynamic therapy, cells from the biofilms were dispersed in a homogenizer and cultured in Brain Heart Infusion broth for quantification of colony-forming units per experimental protocol. For each tested microorganism, two control groups were maintained: one exposed to the laser radiation without the photosensitizer (L+PS-) and other treated with the photosensitizer without exposure to the red laser light (L-PS+). The results were subjected to descriptive statistical analysis.Results: The best results for S. aureus and E. coli biofilms were obtained with photosensitizer concentrations of approximately 300 mu M methylene blue, with microbial reductions of 0.8-1.0 log(10); 150 mu M toluidine blue, with microbial reductions of 0.9-1.0 log(10); and 3000 mu M malachite green, with microbial reductions of 1.6-4.0 log(10).Conclusion: Greater microbial reduction was achieved with the malachite green photosensitizer when used at higher concentrations than those employed for the phenothiazinic dyes. (C) 2011 Elsevier Ltd. All rights reserved.