Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

Pitch discrimination in optimal and suboptimal acoustic environments: electroencephalographic, magnetoencephalographic, and behavioral evidence

Pitch discrimination is a fundamental property of the human auditory system. Our understanding of pitch-discrimination mechanisms is important from both theoretical and clinical perspectives. The discrimination of spectrally complex sounds is crucial in the processing of music and speech. Current methods of cognitive neuroscience can track the brain processes underlying sound processing either with precise temporal (EEG and MEG) or spatial resolution (PET and fMRI). A combination of different techniques is therefore required in contemporary auditory research. One of the problems in comparing the EEG/MEG and fMRI methods, however, is the fMRI acoustic noise. In the present thesis, EEG and MEG in combination with behavioral techniques were used, first, to define the ERP correlates of automatic pitch discrimination across a wide frequency range in adults and neonates and, second, they were used to determine the effect of recorded acoustic fMRI noise on those adult ERP and ERF correlates during passive and active pitch discrimination. Pure tones and complex 3-harmonic sounds served as stimuli in the oddball and matching-to-sample paradigms. The results suggest that pitch discrimination in adults, as reflected by MMN latency, is most accurate in the 1000-2000 Hz frequency range, and that pitch discrimination is facilitated further by adding harmonics to the fundamental frequency. Newborn infants are able to discriminate a 20% frequency change in the 250-4000 Hz frequency range, whereas the discrimination of a 5% frequency change was unconfirmed. Furthermore, the effect of the fMRI gradient noise on the automatic processing of pitch change was more prominent for tones with frequencies exceeding 500 Hz, overlapping with the spectral maximum of the noise. When the fundamental frequency of the tones was lower than the spectral maximum of the noise, fMRI noise had no effect on MMN and P3a, whereas the noise delayed and suppressed N1 and exogenous N2. Noise also suppressed the N1 amplitude in a matching-to-sample working memory task. However, the task-related difference observed in the N1 component, suggesting a functional dissociation between the processing of spatial and non-spatial auditory information, was partially preserved in the noise condition. Noise hampered feature coding mechanisms more than it hampered the mechanisms of change detection, involuntary attention, and the segregation of the spatial and non-spatial domains of working-memory. The data presented in the thesis can be used to develop clinical ERP-based frequency-discrimination protocols and combined EEG and fMRI experimental paradigms.

High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study

Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays.

Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA

The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication. However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the single-stranded DNA or by a direct physical interaction between the DnaB and the DnaG proteins. In this paper we present evidence supporting direct interaction between the two proteins. We have mapped the surfaces of interaction on both DnaG and DnaB and show further that mutations that reduce the physical interaction also cause a significant reduction in primer synthesis. Thus, the physical interaction reported here appears to be physiologically significant.

Selenium and its effects on growth, yield and tuber quality in potato

Selenium (Se) has been demonstrated to be an essential trace element for maintenance of animal and human health. Although it has not been confirmed to be an essential micronutrient in higher plants, there is increasing evidence that Se functions as an antioxidant in plants. Selenium has been shown to exert a beneficial effect on crop growth and promotes stress tolerance at low concentrations. However, the specific physiological mechanisms that underlie the positive effects of Se in plants have not been clearly elucidated. The aims of this study were to determine the Se concentration in potato (Solanum tuberosum L.) and the effects of Se on the accumulation of carbohydrates, growth and yield in potato plants. An additional aim was to study the impact of Se on the total glycoalkaloid concentration in immature potato tubers. The distribution of Se in different biochemical Se fractions and the effect of storage on the Se concentration were studied in Se-enriched tubers. Furthermore, the effect of Se on raw darkening and translocation of Se from seed tubers to the next tuber generation was investigated. Due to the established anti-ageing properties of Se, it was of interest to study if Se affects physiological age and growth vigour of seed tubers. The Se concentrations in the upper leaves, roots, stolons and tubers of potato increased with increasing Se supplementation. The highest Se concentration was reached in young upper leaves, roots and stolons, indicating that added selenate was efficiently utilized and taken up at an early stage. During the growing period the Se concentration declined in the aerial parts, roots and stolons of potato plants whereas an intensive accumulation took place in immature and mature tubers. Selenium increased carbohydrate accumulation in the young upper leaves and in stolons, roots and tubers at maturity. This could not be explained by increased production of photoassimilates as net photosynthesis did not differ among Se treatments. The Se treated plants produced higher tuber yields than control plants, and at the highest Se concentration (0.3 mg kg-1) lower numbers of larger tubers were harvested. Increased yield of Se treated plants suggested that Se may enhance the allocation of photoassimilates for tuber growth, acting as a strong sink for both Se and for carbohydrates. Similarly as for other plant species, the positive impact of Se on the yield of potato plants could be related to its antioxidative effect in delaying senescence. The highest Se supplementation (0.9 mg kg-1) slightly decreased the glycoalkaloid concentration of immature tubers. However, at this level the Se concentration in tubers was about 20 µg g-1 DW. A 100 g consumption of potato would provide about 500 mg of Se, which exceeds the upper safe intake level of 400 µg per day for human dietary. The low Se applications (0.0035 and 0.1 mg kg-1) diminished and retarded the degree of raw darkening in tubers stored for one and eight months, which can be attributed to the antioxidative properties of Se. The storage for 1 to 12 months did not affect the Se concentrations of tubers. In the Se enriched tubers Se was allocated to the organic Se fraction, indicating that it was incorporated into organic compounds in tubers. Elevated Se concentration in the next-generation tubers produced by the Se enriched seed tubers indicated that Se could be translocated from the seed tubers to the progeny. In the seed tubers stored for 8 months, at high levels, Se had some positive effects on the growth vigour of sprouts, but Se had no consistent effect on the growth vigour of seed tubers of optimal physiological age. These results indicate that Se is a beneficial trace element in potato plants that exerts a positive effect on yield formation and improves the processing and storage quality of table potato tubers. These positive effects of Se are, however, dependent on the Se concentration and the age of the potato plant and tuber.

LQ optimal and risk-sensitive control for vehicle suspensions

This paper is concerned with time-domain optimal control of active suspensions. The optimal control problem formulation has been generalised by incorporating both road disturbances (ride quality) and a representation of driver inputs (handling quality) into the optimal control formulation. A regular optimal control problem as well as a risk-sensitive exponential optimal control performance index is considered. Emphasis has been given to practical considerations including the issue of state estimation in the presence of load disturbances (driver inputs). © 2012 IEEE.

Dynamic pricing models for used products in remanufacturing with lost-sales and uncertain quality

In this paper, we investigate the remanufacturing problem of pricing single-class used products (cores) in the face of random price-dependent returns and random demand. Specifically, we propose a dynamic pricing policy for the cores and then model the problem as a continuous-time Markov decision process. Our models are designed to address three objectives: finite horizon total cost minimization, infinite horizon discounted cost, and average cost minimization. Besides proving optimal policy uniqueness and establishing monotonicity results for the infinite horizon problem, we also characterize the structures of the optimal policies, which can greatly simplify the computational procedure. Finally, we use computational examples to assess the impacts of specific parameters on optimal price and reveal the benefits of a dynamic pricing policy. © 2013 Elsevier B.V. All rights reserved.

Differential effects of temperature on fruit development and bean quality of contrasting genotypes of cacao (Theobroma cacao)

The effects of temperature and light integral on fruit growth and development of five cacao genotypes (Amelonado, AMAZ 15/15, SCA 6, SPEC 54/1 and UF 676) were studied in semi-controlled environment glasshouses in which the thermal regimes of cacao-growing regions of Brazil, Ghana and Malaysia were simulated. Fruit losses because of physiological will (cherelle will) were greater at higher temperatures and also differed significantly between genotypes, reflecting genetic differences in competition for assimilates between vegetative and reproductive components. Short-term measurements of fruit growth indicated faster growth rates at higher temperatures. In addition, a significant negative linear relationship between temperature and development time was observed. There was an effect of genotype on this relationship, such that time to fruit maturation at a given temperature was greatest for the clone UF 676 and least for AMAZ 15/15. Analysis of base temperatures, derived from these relationships indicated genetic variability in sensitivity of cacao fruit growth to temperature (base temperatures ranged from 7.5 degrees C for Amelonado and AMAZ 15/15 to 12.9 for SPEC 54/1). Final fruit size was a positive function of beam number for all genotypes and a positive function of light integral for Amelonado in the Malaysia simulated environment (where the temperature was almost constant). In simulated environments where temperature was the main variable (Brazil and Ghana) increases in temperature resulted in a significant decrease in final pod size for one genotype (Amelonado) in Brazil and for two genotypes (SPEC 54/1 and UF 676) in Ghana. It was hypothesised that pod growth duration (mediated by temperature), assimilation and beam number are all determinants of final pod size but that under specific conditions one of these factors may override the others. There was variability between genotypes in the response of beam size and beam lipid content to temperature. Negative relationships between temperature and bean size were found for Amelonado and UF 676. Lipid concentration was a curvilinear function of temperature for Amelonado and UF 676, with optimal temperatures of 23 degrees C and 24 degrees C, respectively. The variability observed here of different cacao genotypes to temperature highlights the need and opportunities for appropriate matching of planting material with local environments.

Feedback of personal retinal images appears to have a motivational impact in people with non-proliferative diabetic retinopathy and suboptimal HbA1c: findings of a pilot study

AIM:
To conduct a pilot study to explore the potential impact of visual feedback of personal retinal images on diabetes outcomes.

METHODS:
Twenty-five participants with non-proliferative diabetic retinopathy and suboptimal HbA1c (> 53 mmol/mol; > 7%) were randomized to receive visual feedback of their own retinal images or to a control group. At baseline and 3-month follow-up, HbA1c, standard measures of beliefs, diabetes-related distress and self-care activities were assessed.

RESULTS:
In unadjusted models, relative to controls, the intervention group showed significantly greater improvement in HbA1c at 3-month follow-up (–0.6% vs. +0.3%, P < 0.01), as well as enhanced motivation to improve blood glucose management (P < 0.05).

CONCLUSIONS:
This small pilot study provides preliminary evidence that visual feedback of personal retinal images may offer a practical educational strategy for clinicians in eye care services to improve diabetes outcomes in non-target compliant patients. A fully powered randomized controlled trial is required to confirm these findings and determine the optimal use of feedback to produce sustained effects.

Optimal and sub-optimal control in Dengue epidemics

This work concerns the application of the optimal control theory to Dengue epidemics. The dynamics of this insect-borne disease is modelled as a set of non-linear ordinary differential equations including the effect of educational campaigns organized to motivate the population to break the reproduction cycle of the mosquitoes by avoiding the accumulation of still water in open-air recipients. The cost functional is such that it reflects a compromise between actual financial spending (in insecticides and educational campaigns) and the population health (which can be objectively measured in terms of, for instance, treatment costs and loss of productivity). The optimal control problem is solved numerically using a multiple shooting method. However, the optimal control policy is difficult to implement by the health authorities because it is not practical to adjust the investment rate continuously in time. Therefore, a suboptimal control policy is computed assuming, as the admissible set, only those controls which are piecewise constant. The performance achieved by the optimal control and the sub-optimal control policies are compared with the cases of control using only insecticides when Breteau Index is greater or equal to 5 and the case of no-control. The results show that the sub-optimal policy yields a substantial reduction in the cost, in terms of the proposed functional, and is only slightly inferior to the optimal control policy. Copyright (C) 2001 John Wiley & Sons, Ltd.

Stream fish, water and habitat quality in a pasture dominated basin, southeastern Brazil

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

Effect of natural antimicrobials on microbiological and sensorial quality of fresh-cut cantaloupe melon

The effect of different natural antimicrobials on the microbiological and sensorial quality of fresh-cut Cantaloupe melons stored up to 10 days at 5°C was examined. Pieces of melon were washed for 1 min at 5ºC in water (control), vanillin (1000 mg/L and 2000 mg/L) or cinnamic acid (148.16 mg/L and 296.32 mg/L). Other antimicrobial treatments consisted of packaging the pieces of melon with an antimicrobial pad which contained cinnamic acid (148.16 mg/L and 296.32 mg/L). After 10 days of storage, significant differences among antimicrobials treatments and water treatment were found. In water treatment, the psychrotroph load was 3.63 ± 0.09 log cfu g-1 meanwhile on all antimicrobial treatments the values ranged from 3.04 ±0.13 log cfu g-1 to 3.28±0.1 log cfu g-1. Mesophilic growth in the control treatment averaged 6.79±0.06 log cfu g-1 meanwhile on antimicrobial treatments the counts were from 5.15±0.01 log cfu g-1 to 5.30±0.03 log cfu g-1. Total coliform levels were 7.8±0.1 log cfu g-1 when melon was washed in water, followed by washing with cinnamon (296.32 mg/L) at 6.5 log cfu g-1 and for the rest of the treatments were around 5.5 log cfu g-1. The treatments did not display differences among mould and yeast growth after 10 days of storage. The sensorial quality decreased throughout storage. However, at the end of storage, the scores ranged between 6.5 and 7, above the minimum level for marketability (level 5). Sensorial panelist noted a ‘sweet’ taste when vanillin was used as sanitizer. In all antimicrobial treatments, no relation was found between a higher dose and a higher microbial reduction. So, vanillin at 1000 mg/L in water or cinnamic acid at 148.16 mg/L provided in water dip or as a pad inside the trays could be optimal natural sanitizers to substitute the use of chlorine in fresh-cut products as Cantaloupe melon.

Survivable Lightpath Provisioning in WDM Mesh Networks Under Shared Path Protection and Signal Quality Constraints

This paper addresses the problem of survivable lightpath provisioning in wavelength-division-multiplexing (WDM) mesh networks, taking into consideration optical-layer protection and some realistic optical signal quality constraints. The investigated networks use sparsely placed optical–electrical–optical (O/E/O) modules for regeneration and wavelength conversion. Given a fixed network topology with a number of sparsely placed O/E/O modules and a set of connection requests, a pair of link-disjoint lightpaths is established for each connection. Due to physical impairments and wavelength continuity, both the working and protection lightpaths need to be regenerated at some intermediate nodes to overcome signal quality degradation and wavelength contention. In the present paper, resource-efficient provisioning solutions are achieved with the objective of maximizing resource sharing. The authors propose a resource-sharing scheme that supports three kinds of resource-sharing scenarios, including a conventional wavelength-link sharing scenario, which shares wavelength links between protection lightpaths, and two new scenarios, which share O/E/O modules between protection lightpaths and between working and protection lightpaths. An integer linear programming (ILP)-based solution approach is used to find optimal solutions. The authors also propose a local optimization heuristic approach and a tabu search heuristic approach to solve this problem for real-world, large mesh networks. Numerical results show that our solution approaches work well under a variety of network settings and achieves a high level of resource-sharing rates (over 60% for O/E/O modules and over 30% for wavelength links), which translate into great savings in network costs.