The geneticvariability induced by in vitro conditions known as somaclonal variation is of practical interest due to its potential uses in plant breeding but, on the other hand, if clonal propagation or transformation is main goal, it becomes an unwelcome phenomenon. Thus, it is important to know frequency, the genomic distribution, the mechanisms and factors influencing somaclonal variation. We studied variabilityof PCR- based DNA markers ofculturedtissuesand regenerated plants of maize and bread wheat. The original A188 line of maize and the somaclones obtained were tested using 38 RAPD and 10 ISSR primers. None of the A188 plants showed variation in the RAPD and ISSR spectra for any of the primers used. However, the PCR spectra obtained from the somaclones demonstrated some variations, i.e., 22 RAPD primers and 6 ISSR primers differentiated at least one somaclonal variant from the progenitor line. Six SCAR markers were developed based on several RAPD and ISSR fragments. The inheritance of these SCAR markers was verified in the selfing progeny of each somaclone in the R 1 –R 4 generations and in the hybrids, with A188 as the
However, as powerful and sensitive as qRT-PCR has the potential to be, it is not without its pitfalls. A fundamental constraint on the accurate interpretation of qRT-PCR data is the necessity of a stable, constitutively-expressed reference gene which can be used as a standard measure for comparing one sample to another . This normalization is typically performed in order to compensate for the variability between samples contributed by the multitude of factors which can influence the cycle threshold (CT) value obtained from a qRT-PCR reaction. Ideal reference genes for the normalization of qRT-PCR data are genes which demonstrate a consistent level of expression among control and treated samples, and in the context of studies under stresses, the expression levels of these reference genes would be unaffected by the stress treatments applied and would allow for the accurate normalization of target gene expression levels between samples. The changes of gene expression levels induced by abiotic stresses like drought, high salinity and cold can be complex and multifaceted, often affecting the expression levels of genes which would otherwise be stable and suitable as reference genes in other experimental circumstances. Consequently, it is crucial that the expression stability of potential reference gene be confirmed under abiotic stressconditions before they are utilized for the normalization of qRT-PCR data generated in these studies. For soybean, several reference genes have been available; however, these genes were validated only undernormal or biotic stressconditions [44–46]. An extended search in literature identified an article on the journal of Pesquisa Agropecua´ria Brasileira by Stolf-Moreira and coworkers which described the search for reference genes for use in qRT-PCR analysis of soybean under drought condition only . Therefore, the primary objective of this study was to determine in systematic manner reference genes which demonstrate a high degree of expression stability under various abiotic stressconditions in soybean, to facilitate a more accurate normalization of qRT-PCR assays.
Tissue-specific and development stage-related expression data are useful in the identification of genes that are involved in defining the precise nature of individual tissues in a given developmental stage. Moreover, the mechanisms controlling the response to drought stress may be associated with root- and/or shoot-related traits. For instance, suppression of shoot growth and/or promotion of primary root growth are considered morphological adjustments enabling plants to adapt better to drought stress [1,3,11,12]. CKs are well-known to positively regulate shoot growth but negatively regulate root growth. As a result, the appropriate control of shoot- and root-related morphological traits, via the modulation of endogenous CK levels prior to the occurrence of a stress, as a preventive measure, is a promising approach for developing economically important drought-tolerant crops [12,32]. Apart from their biochemical characteristics, tissue-specific and development stage-related expression of the CK metabolic genes indicate their functional specification and potential utility for the genetic engineering of specific traits. A well-known example is that among the seven Arabidopsis ATP/ADP IPT genes, IPT1, 3, 5 and 7 are expressed in the vegetative phase and IPT4, 6 and 8 are not. Thus, the ipt1,3,5,7 quadruple mutant has reduced active CK levels which results in morphological adjustment (shorter shoot and longer primary root), hypersensitivity to ABA and enhanced cell membrane integrity contributing to enhanced drought-tolerant phenotypes [11,19].
Such increase in levels of hydrophilic antioxidants, GSH, ascorbic acid and proline under temperature stress, and their reduction under salt stress (except a slight increase in proline) suggested that horse gram expresses a more efficient antioxidant response to high temperatures than for salt stress (Table 1). Ascorbic acid in association with other components of the antioxidant system protects plants against oxidative damage resulting from aerobic metabolism, photosynthesis and a range of pollutants (Waheed et al., 2007). Enhanced production of ascorbic acid and GSH has been correlated to reduced ROS production under temperature stress (Xu et al., 2006). Similarly, increased levels of these two hydrophilic antioxidants in horse gram support the fact that GSH and ascorbate protect the plant against oxidative damage.
Abstract: Characterization of polymeric materials mechanical behavior requires some previous knowledge about their structure, which allows the choice of more appropriated models and methods. Polymeric materials, below their glass transition temperature (T g ), may be handled as perfect elastic solids, allowing the use of classic mechanics to characterize their behavior. Polymers above their T g present a viscous contribution to mechanical behavior, which has to be taken into consideration by modeling it. Adhesively bonded joint, joining of different materials using a polymer as adhesive, adds to the mentioned requirements more parameters, such as surface roughness, adhesive thickness and different types of contributions to adhesively bonded joint strength. This work has the purpose of presenting a mechanical behavior characterization of adhesive bonded joints, concerning their average stress at rupture. A modified Arcan´s device was used to obtain the average stress at rupture under different angles or loading conditions, such as pure shear 0°, pure tensile strength 90° and combined conditions. The experimental results were applied to a theoretical model, which takes into consideration the hydrostatic contribution to the mechanical behavior, called Drucker-Prager Model, which was initially developed to characterize soils.
JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when culturedunder neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro . These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow- derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.
We cannot distinguish whether facilitative belowground inter- actions enhanced plant performance due to niche complementary (different root depth or seasonality between interacting plants) or direct facilitation. All species were perennial, evergreen (except Anthyllis, a facultative summer-deciduous) and had similar growth seasonality. Pot size avoided big differences in spatial distribution of roots. Both facts suggest that niche complementary was not important in our experiment, and that the main process was probably due to direct root effects through exudates and release of secondary compounds. We found, for example, that legumes facilitated neighbour performance. Facilitation of legumes is often mediated by litter, but our experimental time span was too short to allow this process to be important. Recently, Ayres et al.  showed that nitrogen fixed by legumes can be directly transferred Figure 2. Root-to-shoot ratio of individuals of target species (name inside the panel) growing with individuals of the same (solid column) or different species (grey column). P is the significance of ANOVA (ns: non-significant differences). The continuous horizontal line shows mean values of R:S ratio for control plants and dashed lines represent 61 SE. Bars with different letters are significantly different at P,0.05. Asterisk denotes significant differences with control plants. Legend of species is as in Table 1.
The influence of lairage time in a climatized holding area can be explained by the energetic load consumption of birds understress situation (Mendes, 2001; Savenije et al., 2002; Olanrewaju et al., 2006). The birds showed an accentuated consumption of the energetic load in the first 30 minutes of exposition to a stressor factor, resulting thereafter in an adaptation (Bressan & Beraquet, 2002). For short distance, the longer lairage time in the climatized area resulted in the return to the initial thermal comfort condition, increasing chances of survival of the flock. In other words, the thermal stressof broilers might be reversible with the climatization during lairage, because they were still sensible to the climatized environment. For longer journeys, the birds exceeded this critical period, reaching the irreversibility due to the depletion of energetic reserves, being shorter the effect of climatization on the returning to the initial thermal comfort of broilers. The long waiting of weak birds during lairage resulted in an increase of the number of dead birds in this time interval. Therefore, the efficiency of climatization is important when it considers the need of reducing thermal stressof broilers, and the distance showed a direct relation to the effects of lairage time, in terms of rectal temperature and mortality.
. This study was carried out in Rio Claro, SP, Brazil, in a 12 m × 115 m house divided into three equal sections (East, center and West), East-West oriented, and virtually divided on 132 cells, each one measuring 3.0 m × 3.5 m. At the geometric center of each cell the following variables were monitored: dry bulb temperature, relative humidity, air velocity, noise level and light intensity. Average broiler mortality was recorded in each of the three sections. Data collection was made systematically from West to East, opposite to the air flow produced by the tunnel ventilation system, during the warmest period of the day. Measurements took place during the sixth week of production. A geostatistics software tool was used to build spatial distribution maps of the recorded variables in order to infer intermediate stressconditions. It was concluded that the stress zones were located at both ends of the house and the highest mortality index was found at the West sector where the exhaust fans were placed.
We also examined whether BLI could sense the increase in b- cell mass in HFD-fed mice. Ins1-luc BAC transgenic male mice fed either a RD (n = 4) or an HFD (n = 6) were imaged at 4, 8, and 12 weeks (Figure 5A). The bioluminescence intensity was significantly enhanced in the HFD-fed mice at 8 weeks (RD: 1.9760.80610 6 photons/sec; HFD: 7.4261.52610 6 photons/sec; P = 0.022) and accompanied by an increase in the body fat ratio (RD: 7.862.9%; HFD: 27.965.4%; P = 0.019) and in FBG levels (RD: 125628 mg/dL; HFD: 271635 mg/dL; P = 0.018) (Figure 5B– E). However, although the rate of increase in the signal after 8 weeks was substantially higher in the HFD-fed mice than in the RD-fed mice (379%), the rate of increase in b-cell mass differed slightly (121%; RD: 1.3160.28 mg; HFD: 1.5960.26 mg; P = 0.49) (Figure 5F). Immunohistochemical analysis of islets from HFD-fed mice showed a similar pattern to control islets in insulin and luciferase reactivity, so that we could not find a clear reason for the discrepancy in the rate of increase between the BLI signal and b-cell mass (Figure 5G) Interestingly, some mice, especially HFD-treated mice, developed dispersed luminescence from the central to right abdominal regions as shown in Figure 5, probably owing to the signal emission from the duodenum lobe that is ventrally covered by the liver and undetectable in MIP-Luc-VU mice.
The use of mineral fertilizers on agricultural crops, including maize, is a practice that causes environmental damage and economical losses. A promising alternative, to improve productivity and reduce fertilizer use is the use of benefical microrganisms associated with plants, particulary the growth-promoting rhizobacteria. These bacteria live in the rhizosphere and are capable of colonizing different planttissues, benefiting plant growth through mechanisms of growth promotion. In the search for sustainable and more profitable alternative, this study aimed to isolate, characterize, monitor and select rhizobacteria associated with Amazonian guarana that possessed characteristics ofplant growth promoters for use as inoculants in maize seeds. Rhizosphere soil samples from five guarana plants were collected and the isolation of rhizobacteria was performed. Molecular characterization was performed by sequencing the 16S rDNA for analysis of microbial diversity and identification of strains. It was evaluated the ability of strains to produce indole acetic acid (IAA), fix atmospheric nitrogen, solubilize inorganic phosphate and produce siderophores. The analysis of microbial diversity indicated similarity between the bacterial community isolated from the rhizosphere of guarana and that found in the literature to maize. It was observed predominance of Proteobacteria phylum, being mostly represented by the genus Burkholderia. Of the total 101 strains obtained, 89% were able to produce IAA, 23% fixed atmospheric nitrogen, 43% solubilized inorganic phosphate and 24% produced siderophores. Five strains were selected for testing growth promotion of maize under greenhouse conditions; these were identified by complete sequencing of the 16S rDNA and compose the treatments as follows: RZ2MS9 - Bacillus sp. (T1), RZ2MS16 - Burkholderia ambifaria (T2) and
Water permeation through aquaporins is a passive process that follows the direction of osmotic pressure across the membrane. Although many aquaporins function as always-open channels, a subgroup of aquaporins, particularly in plants have evolved a sophisticated molecular mechanism through which the channel can be closed in response to harsh conditionsof the environment, under which exchange of water can be harmful for the organism (Törnroth-Horsefield et al., 2005). Plants respond to drought or flood conditions by shutting down almost all of their AQP .In humans, 13 different AQP (AQP0–AQP12) have been characterized in various organs (kidneys, eyes, and the brain). The solved structures of several AQP at high resolution are indicative of a conserved protein architecture in the whole family (Wang and Tajkhorshid, 2007).
Figure 1. A: The irst step in contrast vision is how it arises from the objective world. In this example, sine wave luminance gratings were generated on the screen of a CRT monitor. This type of system is able to represent the luminance modulation of sine wave gratings across a wide range of spatial frequencies that encompass the human visible range. At high spatial frequencies, a progressive contrast loss occurs, which quickly sets the useful range for the modulation of representations on a CRT display. Different physical principles work with different display types, but generally the effect is the same: a low-pass MTF. At low spatial frequencies, the MTF is usually limited only by the display size. B: The MTF for the eye optical system taken as a single entity despite the fact that it is composed of several optical elements with different properties. The eye optics MTF is recorded at the eye fundus. It has a low-pass shape, similar to the CRT display MTF, but it transmits a shorter range of high spatial frequencies than it is possible to represent in different displays available to the experimenter. C: Contrast transfer of the entire visual system in a human subject obtained using psychophysical methods. The CSF is the result of contrast transfer through a series of elements organized in a cascade that comprise the generation of sine wave gratings on a CRT monitor, high spatial frequency attenuation by the eye optical elements, and both low and high spatial frequency contrast attenuation by neural circuits. As a consequence, the visual system CSF is band-pass.
Anthracnose is an important disease of bell pepper (Capsicum annuum) in Brazil and worldwide. This research investigated the efficacy ofplant extracts for control of this disease in bell pepper fruits caused by Colletotrichum acutatum under controlled conditionsand analyzed the best treatments in relation to different extract and inoculum concentrations, pathogen isolates, and temperatures. In the preliminary screening, among aqueous or ethanolic extracts of 16 plant species evaluated, the best were 6% aqueous garlic, mallow, and ginger extracts, which reduced disease severity by more than 97%. Garlic extract required the highest concentrations to reduce severity by 75% (CL 75 ) when compared to ginger and mallow extracts, but exhibited the best efficacy in relation to different inoculum concentrations and isolates of C. acutatum. It was also able to maintain greater than 90% efficacy at 20-30 o C. The garlic aqueous
In this contribution, basic definitions and concepts ofstress in plants are summarised. Furthermore, the general adaptation syndrome as a response to various types of environmental stress is considered. Finally, plant growth under optimum conditions as opposed to that understressconditions is evaluated. ”Stress is the nonspecific response of the body to any demand”…..”Confusion between stress as both an agent and a result can be avoided only by the distinction between ‘stress’ and ‘stressor’.” (S ELYE , 1973). A stressed plant decreases its activity or growth (O SMOND et al., 1987), but the usefulness of unfavourable conditions for plant survival was generally accepted. For example L ARCHER (1987) has stated that ”Stress ist ein notwendiger Trainingsanreiz im Leben”. In fact, no plant is able to perform its whole living cycle without experiencing multiple unfavourable environmental effects (S EELEY , 1990). In an extensive review, L ESHEM & K UIPER (1996) analysed whether there is a general adaptation syndrome response to various types of environmental stress in plants. They dealt with several environmental stress categories, i.e. heat, drought, salinity, chilling, freezing, oxidative stress, desiccation, ozone and UV-B radiation. All these stressors seem to manifest similar endogenous ”pleiotropic general adaptation syndrome responses whose final expression includes the concerted production” of oxy-free radical scavengers and anti-oxidants, osmoregulants, abscisic acid, jasmonates, nitric oxide, heat shock proteins and phytochelatins. On the other hand, e.g. P ALTA (1990) emphasised the general importance of changes of the membrane properties. According to his measurements, early events responding to unfavourable environmental factors at the cellular level include functional and structural changes in the membrane. Nevertheless, the problem of the general adaptation syndrome response might be more complicated. L AKSO (1990) underlined the importance of the plant organisational level and both morphological and anatomical variations that play an important role in the relationship between the environment including its adverse effects and resulting physiological responses. A plant may react differently according to the hierarchical level of the performed study. Also, plant responses to an environmental stress may be modified by a range of other environmental factors both prior and during the experiment.
At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readi- ness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs) that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i) exosomes act as carriers of signals in utero-placental compartments and ii) exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to deter- mine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC). We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor pla- centae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exo- somes isolated by differential centrifugations. Both growth conditions (normaland oxidative stress induced) produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H) 3, heat shock protein (HSP) 70 and activated form of pro- senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK) (P-p38 MAPK) co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress condi- tions than standard conditions (p<0.05). Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell- to-cell communication. This study determined AEC exosome characteristics and their cargo reflected the physiologic status of the cell of origin and suggests that AEC-derived exoso- mal p38 MAPK plays a major role in determining the fate of pregnancy. Understanding the a11111
Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite’s biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites’ biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents.
obtain a ﬁnal composition of the mobile phase at different time. The low rate and injection volumes were 0.4 mL/ min and 3.5 µL respectively. The analysis was carried out under gradient conditions as follows, time (min)/A (v/v):B (v/v); T0.01/65:35, T3.0/50:50, T5.0/50:50, T10.0/20:80, T11.0/65:35 and T12.0/65:35. The data was acquired at 215 nm for 12 min and processed by use of Empower Pro data handling system. The UPLC runs using the modiﬁed method ended with the required resolution of all peaks along with the good baseline and good signal to noise ratio. The ﬁnal method was then optimized with the above mobile phase conditions with the use of a BEH C18 1.7 µm×2.1 mm x 50 mm Column, keeping the column temperature at 30 °C. Sample temperature was maintained at 5 °C. The degradants were ﬁnally resolved with required resolution.