Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR), genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress), and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity). The raw cycle threshold (Ct) data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M), and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding.

PPARs are members of the nuclear hormone receptor superfamily and are primarily involved in lipid metabolism. The expression patterns of all 3 PPAR isotypes in 22 adult rat organs were analyzed by a quantitative ribonuclease protection assay. The data obtained allowed comparison of the expression of each isotype to the others and provided new insight into the less studied PPAR beta (NR1C2) expression and function. This isotype shows a ubiquitous expression pattern and is the most abundant of the three PPARs in all analyzed tissues except adipose tissue. Its expression is especially high in the digestive tract, in addition to kidney, heart, diaphragm, and esophagus. After an overnight fast, PPAR beta mRNA levels are dramatically down-regulated in liver and kidney by up to 80% and are rapidly restored to control levels upon refeeding. This tight nutritional regulation is independent of the circulating glucocorticoid levels and the presence of PPAR alpha, whose activity is markedly up-regulated in the liver and small intestine during fasting. Finally, PPAR gamma 2 mRNA levels are decreased by 50% during fasting in both white and brown adipose tissue. In conclusion, fasting can strongly influence PPAR expression, but in only a few selected tissues.

Biomechanical stability of a posterior-alone fixation technique after craniovertebral junction realignment.

OBJECTIVE: The aim of the current study was to investigate the biomechanical stability and fixation strength provided by a posterior approach reconstruction technique to realign the craniovertebral junction.¦METHODS: We tested seven human cadaver occipito-cervical spines (occiput-C4) by applying pure moments of ±1.5 Nm on a spine tester. Each specimen was tested in the following modes: 1) intact; 2) injured; 3) spacers alone at C1-C2 articulation (S); 4) spacers plus C1-C2 Posterior Instrumentation (S+PI); and 5) spacers plus C1-C2 posterior instrumentation plus midline wiring (S+PI+MLW). C1-C2 range of motion for each construct was obtained in flexion-extension, lateral bending, and axial rotation.¦RESULTS: In all the loading modes, S, S+PI, and S+PI+MLW constructs significantly reduced range of motion compared with the intact and injured condition (P < 0.05). There was no statistical difference between any of the three instrumentation constructs (P > 0.05).¦CONCLUSIONS: This study investigated the biomechanics of the posterior approach technique for realignment of the craniovertebral junction and also made comparisons with additional posterior fixations. The stand-alone spacers were stable in all three loading modes. Posterior instrumentation increased the stability as compared to stand-alone spacers. The third point of fixation, carried out by using midline wiring, increased the stability further. However, there was not much difference in the stability imparted with the midline wiring versus without. The present study highlights the biomechanics of this novel concept and reaffirms the view that distraction of the C1-C2 articular facets and direct articular joint atlantoaxial fixation would be an ideal method of management of basilar invagination.

Iowa Fen Rapid Assessment Method for Wetlands v. 1 Quantitative Rating, 2016

Rapid assessment methods are valuable tools for collecting information about the quality and status of natural systems. However, they are not a substitute for detailed surveys of those systems. Users of this method should consider that the method may under-score or over-score the site that’s assessed, especially when the site is not a typical fen (i.e. a sedge meadow could score lower than fens, but in-fact be a relatively high quality sedge meadow). This assessment can be used throughout most of the spring, summer and fall period, however the ideal “index period” would be from late May through early October when native plant communities, as well as invasive species, are most apparent.

Holographic optical tweezers combined with back-focal-plane displacement detection

A major problem with holographic optical tweezers (HOTs) is their incompatibility with laser-based position detection methods, such as back-focal-plane interferometry (BFPI). The alternatives generally used with HOTs, like high-speed video tracking, do not offer the same spatial and temporal bandwidths. This has limited the use of this technique in precise quantitative experiments. In this paper, we present an optical trap design that combines digital holography and back-focal-plane displacement detection. We show that, with a particularly simple setup, it is possible to generate a set of multiple holographic traps and an additional static non-holographic trap with orthogonal polarizations and that they can be, therefore, easily separated for measuring positions and forces with the high positional and temporal resolutions of laser-based detection. We prove that measurements from both polarizations contain less than 1% crosstalk and that traps in our setup are harmonic within the typical range. We further tested the instrument in a DNA stretching experiment and we discuss an interesting property of this configuration: the small drift of the differential signal between traps.

Early Iron Age Pottery: A Quantitative Approach. Proceedings of the International Round Table organized by the Swiss School of Archaeology in Greece (Athens, Novembre 28-30, 2008).

Quantitative approaches in ceramology are gaining ground in excavation reports, archaeological publications and thematic studies. Hence, a wide variety of methods are being used depending on the researchers' theoretical premise, the type of material which is examined, the context of discovery and the questions that are addressed. The round table that took place in Athens on November 2008 was intended to offer the participants the opportunity to present a selection of case studies on the basis of which methodological approaches were discussed. The aim was to define a set of guidelines for quantification that would prove to be of use to all researchers. Contents: 1) Introduction (Samuel Verdan); 2) Isthmia and beyond. How can quantification help the analysis of EIA sanctuary deposits? (Catherine Morgan); 3) Approaching aspects of cult practice and ethnicity in Early Iron Age Ephesos using quantitative analysis of a Protogeometric deposit from the Artemision (Michael Kerschner); 4) Development of a ceramic cultic assemblage: Analyzing pottery from Late Helladic IIIC through Late Geometric Kalapodi (Ivonne Kaiser, Laura-Concetta Rizzotto, Sara Strack); 5) 'Erfahrungsbericht' of application of different quantitative methods at Kalapodi (Sara Strack); 6) The Early Iron Age sanctuary at Olympia: counting sherds from the Pelopion excavations (1987-1996) (Birgitta Eder); 7) L'aire du pilier des Rhodiens à Delphes: Essai de quantification du mobilier (Jean-Marc Luce); 8) A new approach in ceramic statistical analyses: Pit 13 on Xeropolis at Lefkandi (David A. Mitchell, Irene S. Lemos); 9) Households and workshops at Early Iron Age Oropos: A quantitative approach of the fine, wheel-made pottery (Vicky Vlachou); 10) Counting sherds at Sindos: Pottery consumption and construction of identities in the Iron Age (Stefanos Gimatzidis); 11) Analyse quantitative du mobilier céramique des fouilles de Xombourgo à Ténos et le cas des supports de caisson (Jean-Sébastien Gros); 12) Defining a typology of pottery from Gortyn: The material from a pottery workshop pit, (Emanuela Santaniello); 13) Quantification of ceramics from Early Iron Age tombs (Antonis Kotsonas); 14) Quantitative analysis of the pottery from the Early Iron Age necropolis of Tsikalario on Naxos (Xenia Charalambidou); 15) Finding the Early Iron Age in field survey: Two case studies from Boeotia and Magnesia (Vladimir Stissi); 16) Pottery quantification: Some guidelines (Samuel Verdan).

Quantitative Guidelines for Thin Maintenance Surfaces, TR-435, 2003

What follows are the refined guidelines from the Thin Maintenance Surface: Phase II Report. For that report, test sections were created and monitored along with some existing test sections. From the monitoring and evaluation of these test sections, literature reviews, and the experience and knowledge of the authors, the following guidelines were created. More information about thin maintenance surfaces and their uses can be found in the above-mentioned report.

Arthro-IRM directe en traction axiale: technique et intérêt [Technique and value of direct MR arthrography applying articular distraction]

Direct MR arthrography has a better diagnostic accuracy than MR imaging alone. However, contrast material is not always homogeneously distributed in the articular space. Lesions of cartilage surfaces or intra-articular soft tissues can thus be misdiagnosed. Concomitant application of axial traction during MR arthrography leads to articular distraction. This enables better distribution of contrast material in the joint and better delineation of intra-articular structures. Therefore, this technique improves detection of cartilage lesions. Moreover, the axial stress applied on articular structures may reveal lesions invisible on MR images without traction. Based on our clinical experience, we believe that this relatively unknown technique is promising and should be further developed.

Quantitative multi-parameter mapping of R1, PD(*), MT, and R2(*) at 3T: a multi-center validation.

Multi-center studies using magnetic resonance imaging facilitate studying small effect sizes, global population variance and rare diseases. The reliability and sensitivity of these multi-center studies crucially depend on the comparability of the data generated at different sites and time points. The level of inter-site comparability is still controversial for conventional anatomical T1-weighted MRI data. Quantitative multi-parameter mapping (MPM) was designed to provide MR parameter measures that are comparable across sites and time points, i.e., 1 mm high-resolution maps of the longitudinal relaxation rate (R1 = 1/T1), effective proton density (PD(*)), magnetization transfer saturation (MT) and effective transverse relaxation rate (R2(*) = 1/T2(*)). MPM was validated at 3T for use in multi-center studies by scanning five volunteers at three different sites. We determined the inter-site bias, inter-site and intra-site coefficient of variation (CoV) for typical morphometric measures [i.e., gray matter (GM) probability maps used in voxel-based morphometry] and the four quantitative parameters. The inter-site bias and CoV were smaller than 3.1 and 8%, respectively, except for the inter-site CoV of R2(*) (<20%). The GM probability maps based on the MT parameter maps had a 14% higher inter-site reproducibility than maps based on conventional T1-weighted images. The low inter-site bias and variance in the parameters and derived GM probability maps confirm the high comparability of the quantitative maps across sites and time points. The reliability, short acquisition time, high resolution and the detailed insights into the brain microstructure provided by MPM makes it an efficient tool for multi-center imaging studies.

Les aquaporines 4 et 9 dans le système nerveux central : expression lors de la différenciation cellulaire et implication dans le métabolisme énergétique astrocytaire "in vitro"

RESUME : Les aquaporines (AQPs) sont des protéines membranaires perméables à l'eau (aquaporines strictes) et, pour certaines d'entre elles, également au glycérol (aquaglycéroporines). Ces protéines sont présentes dans les bactéries, les plantes et les différents organes des mammifères. Dans le cerveau, la moindre augmentation de volume hydrique peut avoir de graves conséquences sur son fonctionnement, d'où l'importance de la régulation de l'homéostasie de l'eau grâce aux AQPs. L'AQP4, une aquaporine stricte, est présente dans les astrocytes et est impliquée dans la formation et la résorption des oedèmes cérébraux. En revanche, l'AQP9 est une aquaglycéroporine, qui est localisée non seulement dans les astrocytes mais également dans les neurones catécholaminergiques. Bien que la distribution de l'AQP4 dans le cerveau soit clairement établie, la présence de l'AQP9 est toujours une donnée controversée et son rôle fonctionnel dans le système nerveux central n'est pas connu. Par ailleurs, aucune donnée n'existe sur l'expression des AQP4 et 9 lors de la différenciation de cellules souches neurales foetales (CSNf) en astrocytes ou en neurones catécholaminergiques. Dans la première partie de ce travail, un protocole a été mis au point permettant de différencier des CSNf de souris en astrocytes et neurones, dont des neurones catécholaminergiques. La caractérisation des cultures de CSNf et des cultures mixtes par immunofluorescence a permis de montrer que l'immunomarquage AQP9 est présent dans les CSNf et est conservé lors de leur différenciation en astrocytes ou en neurones catécholaminergiques. Les résultats obtenus ont mis en évidence une très bonne corrélation entre l'expression de la TH (tyrosine hydroxylase: enzyme limitante de la synthèse des catécholamines) et celle de l'AQP9 lors de la différenciation des CSNf en neurones catécholaminergiques. Par contre, l'immunomarquage AQP4 n'est pas présent dans les CSNf alors qu'il est observé dans les astrocytes. De plus, aucun immunomarquage AQP4 ou AQP9 n'a été observé dans les neurones NIAP2-positifs. Dans la deuxième partie de ce travail, l'expression des AQP4 et 9 a été quantifiée dans les CSNf ainsi que dans trois populations d'astrocytes présentant des propriétés métaboliques différentes. Ces trois populations astrocytaires sont issues de la différenciation des CSNf par le CNTF, le LIF ou le sérum de veau foetal. Les analyses par RTPCR quantitative et western blot ont montré une augmentation de l'expression de l'AQP9 et de l'AQP4 corrélée à l'acquisition de propriétés métaboliques spécifiques des astrocytes matures. Dans la dernière partie, la technique d'ARN interférents a permis d'étudier le rôle fonctionnel de l'AQP9 dans le modèle de culture pure d'astrocytes différenciés par le sérum. L'inhibition de l'expression d'AQP9 entraîne une diminution de la perméabilité au glycérol et une augmentation de l'utilisation de glucose, corrélée à une stimulation du métabolisme oxydatif astrocytaire. En revanche, 1a baisse d'expression d'AQP9 n'a aucun effet sur la glycolyse anaérobie ni sur la libération du lactate. En conclusion, dans ce modèle in vitro, seule l'AQP9 est exprimée dans les CSNf et les neurones catécholaminergiques alors que dans Ies astrocytes, à la fois l'AQP9 et l'AQP4 sont exprimées. Cette distribution est identique à celle observée in vivo et confirme la localisation spécifique de l'AQP9 dans les neurones catécholaminergiques. De plus, ces résultats montrent, pour la première fois, l'implication de l'AQP9 dans la perméabilité des astrocytes au glycérol et son implication dans le métabolisme énergétique astrocytaire. ABSTACT : Aquaporins (AQPs) are membrane proteins permeable to water (orthodoxes aquaporins) and some of them are also permeable to glycerol (aquaglyceroporins). These proteins are widely expressed in bacteria, plants and mammals. AQP water homeostasis regulation in brain is of primary importance as the brain volume cannot increase. AQP4, an orthodoxe aquaporin, is present in astrocytes and seems to be involved in edema formation and resorption. On the other hand, AQP9 is an aquaglyceroporin which is localised not only in astrocytes but also in catecholaminergic neurons. Although AQP4 distribution in brain is clearly established, the presence of AQP9 is still a discussed data and its functional role in the central nervous system is unknown. In addition, no data exists on AQP4 or AQP9 expression during fetal neural stem cells (fNSC) differentiation into astrocytes or catecholaminergic neurons. In the first part of this work, a protocol was developed to differentiate mouse fNSC into astrocytes and neurons, with the aim to obtain catecholaminergic neurons. By immunefluorescence, we have shown that AQP9 is expressed in fNSC cultures and also in astrocytes and catecholaminergic neurons in mixt cultures. The results obtained highlighted a very good correlation between TH expression (tyrosin hydroxylase being a limiting enzyme of catecholamines synthesis) and AQP9 in fNSC and all along their differentiation into catecholaminergic neurons. On the other hand, AQP4 immunolabelling is not observed in fNSC whereas it is in astrocytes. Moreover, neitheir AQP4, nor AQP9 immunoreactivity was observed in MAP2-positive neurons. In the second part of this work, AQP4 and AQP9 expression was quantified in fNSC and in three populations of astrocytes presenting different metabolic properties. These three astrocyte populations result from fNSC differentiation by addition of CNTF, LIF or fetal calf serum. Quantitative RT-PCR and western blot analyses have shown an increase in both AQP4 and AQP9 expression, correlated with the acquisition of specific metabolic properties of mature astrocytes. In the last part, siRNA were used to study the functional role of AQP9 in the pure astrocyte culture model differentiated by addition of fetal calf serum. Inhibition of AQP9 expression leads to a decrease of glycerol uptake and to an increase of glucose uptake, correlated with a stimulation of the astrocyte oxydative metabolism. On the other hand, inhibition of AQP9 expression does not have any effect on anaerobic glycolysis nor on lactate release. In conclusion, in this in vitro model, only AQP9 is expressed in fNSC and in catecholaminergic neurons whereas in astrocytes, both AQP9 and AQP4 are expressed. This distribution is identical to that observed in vivo and confirms the specific AQP9 localization in catecholaminergic neurons. IVloreover, these results show, for the first time, that AQP9 is implicated in glycerol uptake and in astrocyte energetic metabolism. Résumé large public : Les aquaporines, des protéines localisées dans les membranes cellulaires sont, comme leur nom l'indique, des canaux à eau. Pendant longtemps, il a été considéré que l'eau diffusait librement dans et à travers les cellules; la caractérisation des AQPs a révolutionné la vision des scientifiques concernant les mouvements d'eau entre les différents compartiments infra et extracellulaires, et a d'ailleurs valu le Prix Nobel à Peter Agre en 1992. Certaines AQPs, dites "strictes", laissent passer uniquement l'eau et participent au contrôle du volume hydrique. Ce contrôle est particulièrement important pour le bon fonctionnement du cerveau en raison de la présence de la boîte crânienne qui limite les variations de volume. D'autres AQPs, les aquaglycéroporines, sont perméables non seulement à l'eau mais également à d'autres molécules comme le glycérol. Elles facilitent, par exemple, la sortie du glycérol des cellules graisseuses et sa capture par les cellules du foie afin de produire du glucose en période de jeûne. Le cerveau est principalement composé de deux types de cellules: les neurones et les cellules gliales, majoritairement des astrocytes. L'AQP4, une AQP stricte, est présente dans les astrocytes et joue un rôle dans la formation et la résorption des oedèmes cérébraux. L'AQP9, une aquaglycéroporine, est également présente dans les astrocytes et dans une population spécifique de neurones, les neurones catécholaminergiques, touchés dans la maladie de Parkinson. A ce jour, la présence de l'AQP9 dans le cerveau est une donnée controversée et son rôle fonctionnel est inconnu. Ce travail de thèse a permis de montrer que l'AQP9 est bien présente d'une part dans les cellules souches neurales foetales et d'autre ,part dans les astrocytes et neurones catécholaminergiques issus de leur différenciation. De plus, ces expériences ont mis en évidence un rôle de l'AQP9 dans l'entrée du glycérol dans les astrocytes, ce qui pourrait être bénéfique dans des conditions d'ischémie. Enfin, les .résultats de cette étude suggèrent également un rôle de l'AQP9 dans le métabolisme énergétique des astrocytes. L'ensemble de ces travaux démontre le rôle important de l'AQP9 dans le cerveau et ouvre de nouvelles perspectives quant aux rôles des AQPs dans des situations pathologiques telles que l'ischémie cérébrale ou encore la maladie de Parkinson.

Spiral CT aortography: an efficient technique for the diagnosis of traumatic aortic injury.

The objective of this study was to assess the efficiency of spiral CT (SCT) aortography for diagnosing acute aortic lesions in blunt thoracic trauma patients. Between October 1992 and June 1997, 487 SCT scans of the chest were performed on blunt thoracic trauma patients. To assess aortic injury, the following SCT criteria were considered: hemomediastinum, peri-aortic hematoma, irregular aspect of the aortic wall, aortic pseudodiverticulum, intimal flap and traumatic dissection. Aortic injury was diagnosed on 14 SCT examinations (2.9 %), five of the patients having had an additional digital aortography that confirmed the aortic trauma. Twelve subjects underwent surgical repair of the thoracic aorta, which in all but one case confirmed the aortic injury. Two patients died before surgery from severe brain lesions. The aortic blunt lesions were confirmed at autopsy. According to the follow-up of the other 473 patients, we are aware of no false-negative SCT examination. Our limited series shows a sensitivity of 100 % and specificity of 99.8 % of SCT aortography in the diagnosis of aortic injury. It is concluded that SCT aortagraphy is an accurate diagnostic method for the assessment of aortic injury in blunt thoracic trauma patients.

Defining risk thresholds for a 10-year probability of hip fracture model that combines clinical risk factors and quantitative ultrasound: results using the EPISEM cohort.

Using a large prospective cohort of over 12,000 women, we determined 2 thresholds (high risk and low risk of hip fracture) to use in a 10-yr hip fracture probability model that we had previously described, a model combining the heel stiffness index measured by quantitative ultrasound (QUS) and a set of easily determined clinical risk factors (CRFs). The model identified a higher percentage of women with fractures as high risk than a previously reported risk score that combined QUS and CRF. In addition, it categorized women in a way that was quite consistent with the categorization that occurred using dual X-ray absorptiometry (DXA) and the World Health Organization (WHO) classification system; the 2 methods identified similar percentages of women with and without fractures in each of their 3 categories, but the 2 identified only in part the same women. Nevertheless, combining our composite probability model with DXA in a case findings strategy will likely further improve the detection of women at high risk of fragility hip fracture. We conclude that the currently proposed model may be of some use as an alternative to the WHO classification criteria for osteoporosis, at least when access to DXA is limited.

Quality of Learners' Time and Learning Performance Beyond Quantitative Time-on-Task

Peer-reviewed

Genetic diversity of irrigated barley based on molecular and quantitative data and on malting quality

The objective of this work was to quantify the genetic diversity of elite genotypes of irrigated barley in the Brazilian savanna. Thirty elite barley genotypes from Embrapa Cerrados' collection were evaluated using 160 RAPD markers, 12 agronomic traits related to yield components, and 10 malting quality parameters. The genetic dissimilarity matrices based on molecular markers, quantitative traits, and malting quality characters were calculated and a cluster analysis was performed using the unweighted pair-group method with arithmetic mean (UPGMA) as grouping criterion. High genetic diversity among accessions were observed. The estimated genetic dissimilarities were weakly correlated, showing the complementarity of the different character groups. Selection indices and graphical dispersion analysis allowed the selection of promising genotypes and the indication of suitable crosses for maximizing the heterotic effects in breeding programs for irrigated barley in the Brazilian savanna.