Preliminary evaluation of a non-invasive device for monitoring intracranial pressure waveforms in dogs.

OBJECTIVES: The objective of this study was to compare waveforms obtained with a new device for the non-invasive monitoring of intracranial pressure (ICP) in dogs with and without neurological disease. MATERIALS AND METHODS: This prospective study was conducted on both neurologically normal dogs and dogs with neurological diseases. First, non-invasive ICP waveforms were recorded in normal dogs using the Braincare® BcMM 2000 monitor while the dogs were under general anaesthesia induced for procedures unrelated to this study. The dogs were positioned in lateral recumbency, and the sensor was placed over the skin of the parietal region. Secondly, non-invasive ICP waveforms were monitored in dogs with brain and spinal disease until waveforms with characteristic peaks were acquired. All the recorded signals were amplified, filtered and digitalized, by the device, and then transferred to a computer for analysis. RESULTS: Normal pulse waveforms indicating normal brain complacency were observed in eight neurologically normal dogs. In six dogs with brain disease, abnormal pulse waveforms were observed suggesting increased ICP and decreased brain complacency. Four dogs with spinal disease undergoing myelography, had normal waveforms before contrast medium injection and abnormal pulse waveforms during contrast medium injection, indicating a potential increase in ICP. CLINICAL SIGNIFICANCE: Based on these preliminary observations, this method was capable of detecting abnormal pulse waveforms that suggested increased ICP.

A high efficient protocol for soybean root transformation by Agrobacterium rhizogenes and most stable reference genes for RT-qPCR analysis.

KEY MESSAGE: A 55% transformation efficiency was obtained by our optimized protocol; and we showed that GmELF1 - ß and GmELF1 - α are the most stable reference genes for expression analyses under this specific condition. Gene functional analyses are essential to the validation of results obtained from in silico and/or gene-prospecting studies. Genetic transformation methods that yield tissues of transient expression quickly have been of considerable interest to researchers. Agrobacterium rhizogenes-mediated transformation methods, which are employed to generate plants with transformed roots, have proven useful for the study of stress caused by root phytopathogens via gene overexpression and/or silencing. While some protocols have been adapted to soybean plants, transformation efficiencies remain limited; thus, few viable plants are available for performing bioassays. Furthermore, mRNA analyses that employ reverse transcription quantitative polymerase chain reactions (RT-qPCR) require the use of reference genes with stable expression levels across different organs, development steps and treatments. In the present study, an A. rhizogenes-mediated soybean root transformation approach was optimized. The method delivers significantly higher transformation efficiency levels and rates of transformed plant recovery, thus enhancing studies of soybean abiotic conditions or interactions between phytopathogens, such as nematodes. A 55% transformation efficiency was obtained following the addition of an acclimation step that involves hydroponics and different selection processes. The present study also validated the reference genes GmELF1-ß and GmELF1-α as the most stable to be used in RT-qPCR analysis in composite plants, mainly under nematode infection.

Prediction of potential novel microRNAs in soybean when in symbiosis.

MicroRNAs (miRNAs) are small molecules, noncoding proteins that are involved in many biological processes, especially in plants; among these processes is nodulation in the legume. Biological nitrogen fixation is a key process, with critical importance to the soybean crop. This study aimed to identify the potential of novel miRNAs to act during the root nodulation process. We utilized a set of transcripts that were differentially expressed in soybean roots 10 days after inoculation with Bradyrhizobium japonicum, which were obtained in a previous study, and performed a set of computational analyses that led us to select new miRNAs potentially involved in nodulation. Among these analyses, the set of transcripts were submitted to an in silico annotation of noncoding RNAs, including a search of similarity against miRNA public databases, ab initio tools for miRNA identification, structural search against miRNA families, prediction of the secondary structure of miRNA precursors, and prediction of the sequences of mature miRNAs. Subsequently, we applied filter procedures based on miRNA selections described in the literature (e.g., free energy value). In the next step, a manual curation inspection of the annotation was performed and the top candidates were selected and used for prediction of potential target genes, which were later checked manually in the database of the soybean genome. This prediction led us to the identification of 9 potential new miRNAs; among these, 4 were conserved in other plants. Moreover, we predicted their target genes might play important roles in the regulation of nodulation.

Overexpression of the activated form of the AtAREB1 gene (AtAREB1ΔQT) improves soybean responses to water deficit.

Abscisic acid-responsive element binding protein (AREB1) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element motif in the promoter region of ABA-inducible genes. Because AREB1 is not sufficient to direct the expression of downstream genes under non-stress conditions, an activated form of AREB1 (AREB1ΔQT) was created. Several reports claim that plants overexpressing AREB1 or AREB1ΔQT show improved drought tolerance. In our studies, soybean plants overexpressing AREB1ΔQT were characterized molecularly, and the phenotype and drought response of three lines were accessed under greenhouse conditions. Under conditions of water deficit, the transformed plants presented a higher survival rate (100%) than those of their isoline, cultivar BR 16 (40%). Moreover, the transformed plants displayed better water use efficiency and had a higher number of leaves than their isoline. Because the transgenic plants had higher stomatal conductance than its isoline under well-watered conditions, it was suggested that the enhanced drought response of AREB1ΔQT soybean plants might not be associated with altered transpiration rates mediated by ABA-dependent stomatal closure. However, it is possible that the smaller leaf area of the transgenic plants reduced their transpiration and water use, causing delayed stress onset. The difference in the degree of wilting and percentage of survival between the 35S-AREB1ΔQT and wildtype plants may also be related to the regulation of genes that protect against dehydration because metabolic impairment of photosynthesis, deduced by an increasing internal CO2 concentration, was not observed in the transgenic plants.

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions.

Quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O2-depleted environments, such as flooding-stressed plants.

Eficiência nutricional do Nitrogênio e produção de biomassa em Calendula officinalis L. (Asteraceae) em condições de casa de vegetação / Nitrogen nutritional efficiency and biomass production in Calendula officinalis L. (Asteraceae) under greenhouse conditions

O objetivo deste trabalho foi avaliar o efeito da aplicação de diferentes doses de nitrogênio no solo e de épocas de coleta sobre a eficiência nutricional do nitrogênio e a produção de biomassa em Calendula officinalis L. (Asteraceae) em condições de casa de vegetação. O substrato utilizado foi a camada de 0 - 20 cm de Latossolo Vermelho Distroférrico. O delineamento experimental foi inteiramente casualizado, com quatro repetições. Os tratamentos foram constituídos de quatro doses de N (0, 21, 42 e 84mg dm-3) e cinco épocas de colheita (15, 30, 45, 60 e 90 dias após o transplante). O teor de N na inflorescência de calêndula reduziu com as épocas de coleta, redução essa que ocorreu possivelmente devido à menor eficiência da absorção desse elemento pela planta e menor translocação do N para as inflorescências. Pode se concluir que as épocas de avaliação influenciaram na resposta da planta à eficiência de absorção (EFA), sendo a máxima EFA de N aos 51 dias após o transplante das mudas. Observou-se aumento na translocação de N da parte aérea (folhas e caules) para as inflorescências, tendo como consequência a redução do N na parte aérea. Houve incremento na produção de biomassa de calêndula em função das épocas de coleta , e do aumento das doses de N aplicadas no solo. A eficiência nutricional do N foi influenciada pelas épocas de avaliação e pelo teor de N presente no solo.

The aim of this study was to evaluate the effect of both the application of different nitrogen levels on the soil and the harvest periods on nitrogen nutritional efficiency and biomass production in Calendula officinalis L. (Asteraceae) under greenhouse conditions. The employed substrate was 0-20cm layer of Oxisol. Experimental design was completely randomized, with four replicates. Treatments consisted of four N levels (0, 21, 42 and 84 mg dm-3) and five harvest periods (15, 30, 45, 60 and 90 days after transplanting). N levels in Calendula officinalis inflorescences reduced with the harvest periods, and such a reduction was probably due to the lower efficiency of absorption of this element by the plant and to the lower N translocation to inflorescences. We can conclude that the evaluation periods influenced the response of plants to uptake efficiency (UE), and the maximal UE for N was found at 51 days after transplanting of seedlings. There was an increase in N translocation from the shoot (leaves and stems) to inflorescences, which has as consequence N reduction in the shoot. Calendula officinalis biomass production increased with the harvest periods and with the increase in the N levels applied on the soil. N nutritional efficiency was influenced by both the evaluation periods and the N levels present on the soil.

A distinct tymovirus infecting Cassia hoffmannseggii in Brazil.

Leaves of Cassia hoffmannseggii, a wild fabaceous species found in the Atlantic Forest, with a severe mosaic symptom were collected in Pernambuco State, Brazil. By transmission electron microscopy, two types of virus particles were found: the first was recognized as particles of a potyvirus, which was later identified as Cowpea aphid-borne mosaic virus; and the second was isometric and present in high concentration. The observation of vesicles at the periphery of chloroplasts suggested a tymovirus infection, which was confirmed by subsequent assays. A serological assay against several tymovirus antisera resulted in positive reaction of this tymo-like virus with an antiserum of Passion fruit yellow mosaic virus. By means of RT-PCR and using degenerated primers for the conserved region of RNA-dependent RNA polymerase (RdRp) gene of tymoviruses, a specific DNA fragment was amplified and sequenced. Based on this sequence, a specific forward primer was synthesized and successfully used to amplify the 3' terminal genome region, containing the partial RdRp gene and the complete coat protein (CP) sequences. The CP was 188 amino acids (aa) long, and the highest CP aa identity was observed with Kennedya yellow mosaic virus (61 %). Based on the current ICTV demarcation criterion, this isolate was considered as a distinct tymovirus and tentatively named as Cassia yellow mosaic-associated virus.

Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A:AtDREB1A for the improvement of drought tolerance.

We evaluated the molecular, anatomical and physiological properties of a soybean line transformed to improve drought tolerance with an rd29A:AtDREB1A construct. This construct expressed dehydration- responsive element binding protein DREB1A from the stress-inducible rd29A promoter. The greenhouse growth test included four randomized blocks of soybean plants, with each treatment performed in triplicate. Seeds from the non-transformed soybean cultivar BR16 and from the genetically modified soybean P58 line (T(2) generation) were grown at 15% gravimetric humidity for 31 days. To induce water deficit, the humidity was reduced to 5% gravimetric humidity (moderate stress) for 29 days and then to 2.5% gravimetric humidity (severe stress). AtDREB1A gene expression was higher in the genetically modified P58 plants during water deficit, demonstrating transgene stability in T(2) generations and induction of the rd29A promoter. Drought-response genes, including GmPI-PLC, GmSTP, GmGRP, and GmLEA14, were highly expressed in plants submitted to severe stress. Genetically modified plants had higher stomatal conductance and consequently higher photosynthetic and transpiration rates. In addition, they had more chlorophyll. Overexpression of AtDREB1A may contribute to a decrease in leaf thickness; however, a thicker abaxial epidermis was observed. Overexpression of AtDREB1A in soybean appears to enhance drought tolerance.

Transcription factors expressed in soybean roots under drought stress.

To gain insight into stress-responsive gene regulation in soybean plants, we identified consensus sequences that could categorize the transcription factors MYBJ7, BZIP50, C2H2, and NAC2 as members of the gene families myb, bzip, c2h2, and nac, respectively. We also investigated the evolutionary relationship of these transcription factors and analyzed their expression levels under drought stress. The NCBI software was used to find the predicted amino acid sequences of the transcription factors, and the Clustal X software was used to align soybean and other plant species sequences. Phylogenetic trees were built using the Mega 4.1 software by neighbor joining and the degree of confidence test by Bootstrap. Expression level studies were carried out using hydroponic culture; the experiments were designed in completely randomized blocks with three repetitions. The blocks consisted of two genotypes, MG/BR46 Conquista (drought-tolerant) and BR16 (drought-sensitive) and the treatments consisted of increasingly long dehydration periods (0, 25, 50, 75, and 100 min). The transcription factors presented domains and/or conserved regions that characterized them as belonging to the bzip, c2h2, myb, and nac families. Based on the phylogenetic trees, it was found that the myb, bzip and nac genes are closely related to myb78, bzip48 and nac2 of soybean and that c2h2 is closely related to c2h2 of Brassica napus. Expression of all genes was in general increased under drought stress in both genotypes. Major differences between genotypes were due to the lowering of the expression of the mybj7 and c2h2 genes in the drought-tolerant variety at some times. Over-expression or silencing of some of these genes has the potential to increase stress tolerance.

Soybean physiology and gene expression during drought.

Soybean genotypes MG/BR46 (Conquista) and BR16, drought-tolerant and -sensitive, respectively, were compared in terms of morphophysiological and gene-expression responses to water stress during two stages of development. Gene-expression analysis showed differential responses in Gmdreb1a and Gmpip1b mRNA expression within 30 days of water-deficit initiation in MG/BR46 (Conquista) plants. Within 45 days of initiating stress, Gmp5cs and Gmpip1b had relatively higher expression. Initially, BR16 showed increased expression only for Gmdreb1a, and later (45 days) for Gmp5cs, Gmdefensin and Gmpip1b. Only BR16 presented down-regulated expression of genes, such as Gmp5cs and Gmpip1b, 30 days after the onset of moisture stress, and Gmgols after 45 days of stress. The faster perception of water stress in MG/BR46 (Conquista) and the better maintenance of up-regulated gene expression than in the sensitive BR16 genotype imply mechanisms by which the former is better adapted to tolerate moisture deficiency.

Eyelid changes in long-standing leprosy.

To describe eyelid changes in ocular leprosy, 74 patients (148 eyes or 296 eyelids) were examined, focusing on eyelid abnormalities. The adnexal examination included evaluation of the upper eyelid crease pattern, qualitative assessment of the orbicularis oculi muscle function, measurement of the distance between the corneal reflex and the upper eyelid margin (margin reflex distance), and slit-lamp biomicroscopy of the eyelashes and tarsal conjunctiva. Eyelash ptosis was a common finding associated with a multiple upper eyelid crease pattern and trichiasis. In the past, eyelash ptosis has probably been diagnosed as upper eyelid entropion or trichiasis, but in this series entropion was not observed. The distinction between eyelash ptosis, trichiasis, and upper eyelid entropion is important because the surgical management for each is different. Other true leprotic abnormalities of the eyelids are lagophthalmos and lower lid ectropion.

Quantitative study of the effect of dacryocystorhinostomy on lacrimal drainage.

Using scintigraphy, we have studied the lacrimal drainage from the conjunctival sac of normal subjects and patients who have undergone dacryocystorhinostomy. A mathematical model of drainage was constructed that accurately described the complete activity curves for both groups. The initial rate of drainage was shown to be a relevant parameter to characterize drainage, and it was found to be different from normals to patients, indicating that the lacrimal pump mechanism is affected by dacryocystorhinostomy.

Congenital shortening of the anterior lamella of all eyelids: the so-called ablepharon macrostomia syndrome.

The so-called ablepharon macrostomia syndrome is an extremely rate congenital condition that includes abnormal ears, an enlarged, fishlike mouth, absence of lanugo, redundant skin, and vertical shortening of all eyelids. Only four cases have been described so far. In these cases the nature of the eyelid anomalies has not been clearly defined. We report one more case showing that the condition is better described as a severe microblepharon because only the anterior lamella of the eyelids is shortened. The literature about this condition is reviewed, and oculoplastic treatment is discussed.

Palpebral fissure height and downgaze in patients with Graves upper eyelid retraction and congenital blepharoptosis.

PURPOSE: To compare the relation between vertical eyelid fissure height and downgaze in healthy subjects and in patients with Graves upper eyelid retraction and congenital blepharoptosis. METHODS: With a caliper rule, the authors measured the vertical eyelid fissure height of 25 patients with no previous history of eye disease, 34 patients with Graves disease, and 16 patients with congenital blepharoptosis. All measurements were done at the primary position and at four levels of downgaze (10 degrees, 20 degrees, 30 degrees, and 40 degrees). In 13 patients with Graves disease, vertical phoria was quantified with a Maddox rod and prism. RESULTS: The relation between vertical eyelid fissure height and downgaze is linear. The slope was identical for the control subjects and for the patients with Graves disease (b = -1.36) but it was almost zero for the patients with congenital blepharoptosis (b = -0.037). When vertical eyelid fissure height was corrected to arc values, the slope of the relation between vertical eyelid fissure height and downgaze was significantly greater for the patients with Graves disease. Vertical phoria was not related to the amount of Graves upper eyelid retraction. CONCLUSIONS: The data support the hypotheses that upper eyelid retraction secondary to Graves eye disease results from hyperactivity of the levator palpebrae superioris muscle.

Simplifying scleral fixation of posterior chamber intraocular lenses.

We describe a surgical modification for primary scleral fixation of posterior chamber intraocular lenses. The technique keeps intraocular surgical manipulation at a minimum and allows fixation of the lens during accidental rupture of the posterior lens capsule.