DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism.

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Reducing Emergency Medical Service response time via the reallocation of ambulance bases.

The demand for highly efficient and effective services and consumer goods is an essential prerequisite for modern organizations. In healthcare, efficiency and effectiveness mean reducing disabilities and maintaining human life. One challenge is guaranteeing rapid Emergency Medical Service (EMS) response. This study analyzes the EMS of Belo Horizonte, Brazil, using two modeling techniques: optimization and simulation. The optimization model locates ambulance bases and allocates ambulances to those bases. A simulation of this proposed configuration is run to analyze the dynamic behavior of the system. The main assumption is that optimizing the ambulance base locations can improve the system response time. Feasible solutions were found and the current system may be improved while considering economic and operational changes.

Microsatellite loci and genetic structure of artificial populations of Cotesia flavipes (Hymenoptera, Braconidae).

Cotesia flavipes (Cameron) is a parasitoid wasp used in the biological control of the sugarcane borer (Diatraea saccharalis) (Fabr., 1794). Studies on the genetic diversity of C. flavipes are hampered by the lack of highly polymorphic molecular markers. In this report, a set of 11 microsatellite loci were developed from an enriched library of C. flavipes. Four microsatellite loci were polymorphic and were screened in 212 C. flavipes individuals (183 females and 29 males) that were randomly sampled from seven rearing laboratory populations. The number of alleles ranged from two to three. The average inbreeding coefficient (FIS) among all laboratory populations was 0.120, indicating an excess of homozygotes. The average genetic diversity within the laboratory populations was 0.292, which is lower than the values reported for wild Cotesia spp populations. Genetic diversity was most pronounced within laboratory populations (70 to 90%). Most of the observed alleles were fixed or close to fixation. This low overall genetic diversity may have originated from a founder effect, i.e., the contribution of a small number of individuals (genes and alleles) to the formation of these populations. To our knowledge, this study is the first to provide microsatellite loci and an analysis of the genetic structure of C. flavipes. Our results suggest that new introductions of C. flavipes may increase genetic diversity and improve the efficiency of the biological control of D. saccharalis. In addition, population structure data could be used to estimate the minimum number of wasps to be imported.

Paternity identification in sugarcane polycrosses by using microsatellite markers.

Although polycrosses have been used to test the potential of cross-combination of a large number of sugarcane parents, the male parent of the half-sib progenies produced is unknown. The present study aimed to integrate the molecular marker technology to the sugarcane polycross approach by the application of microsatellite markers to identify the male parent of 41 elite clones derived from polycross families. Ten microsatellite [single sequence repeats (SSRs)] primer pairs were used to identify the most likely male parent considering markers present in the selected clone but absent in the female parent. The number of alleles generated by the 10 microsatellite primer pairs ranged from 102 (cross-pollination lantern 4) to 120 (cross-pollination lantern 2) with an average of 113.25 alleles per SSR. The average genetic similarity among the involved parents in the polycrosses was 45.9%. The results of the analysis of the SSR markers absent in the female parent and present only in the selected clone as well as the genetic similarity values allowed the identification of the most likely male parent in 73% of the total clones evaluated and also to detect probable contaminations. The obtained results highlight the importance of using molecular marker technology in the identification and confirmation of the male parent of high-performance clones derived from polycrosses in the sugarcane breeding programs.

Selfing rate estimation in sugarcane under unfavorable natural conditions of crossing by using microsatellite markers.

The self-fertilization or selfing rate estimation using microsatellite markers and its impact on survival and selection rate were evaluated in families derived from polycrosses that involved parents that were widely used in sugarcane breeding in Brazil. These factors were evaluated under unfavorable natural conditions of flowering and crossing. After the germination test, the viable progeny were taken to the field for survival rate evaluation (4, 6, and 10 months) and phenotypic selection at plant cane. The selfing rate estimate based on microsatellite markers present in the progeny and absent in their female parent was 98.5 and 0% for the polycross families derived from IACSP95-5000 and SP89-1115, respectively. The survival and selection rates in the last 2 evaluations were higher for the SP89-1115 outcrossed family than the IACSP95-5000 selfed family. The IACSP95-5000 cultivar excelled either as pollen donor with fertilization capability or viable seed production even under unfavorable natural conditions of crossing. The environment influence (temperature and humidity) had an important role during the polycross.

Analysis of the interaction of antimalarial agents with Plasmodium falciparum glutathione reductase through molecular mechanical calculations.

CONTEXT: Malaria remains a significant global health challenge with emerging resistance to current treatments. Plasmodium falciparum glutathione reductase (PfGR) plays a critical role in the defense mechanisms of malaria parasites against oxidative stress. In this study, we investigate the potential of targeting PfGR with conventional antimalarials and dual drugs combining aminoquinoline derivatives with GR inhibitors, which reveal promising interactions between PfGR and studied drugs. The naphthoquinone Atovaquone demonstrated particularly high affinity and potential dual-mode binding with the enzyme active site and cavity. Furthermore, dual drugs exhibit enhanced binding affinity, suggesting their efficacy in inhibiting PfGR, where the aliphatic ester bond (linker) is essential for effective binding with the enzyme's active site. Overall, this research provides important insights into the interactions between antimalarial agents and PfGR and encourages further exploration of its role in the mechanisms of action of antimalarials, including dual drugs, to enhance antiparasitic efficacy. METHODS: The drugs were tested as PfGR potential inhibitors via molecular docking on AutoDock 4, which was performed based on the preoptimized structures in HF/3-21G-PCM level of theory on ORCA 5. Drug-receptor systems with the most promising binding affinities were then studied with a molecular dynamic's simulation on AMBER 16. The molecular dynamics simulations were performed with a 100 ns NPT ensemble employing GAFF2 forcefield in the temperature of 310 K, integration time step of 2 fs, and non-bond cutoff distance of 6.0 Å.

A mixed model QTL analysis for sugarcane multiple-harvest-location trial data.

Sugarcane-breeding programs take at least 12 years to develop new commercial cultivars. Molecular markers offer a possibility to study the genetic architecture of quantitative traits in sugarcane, and they may be used in marker-assisted selection to speed up artificial selection. Although the performance of sugarcane progenies in breeding programs are commonly evaluated across a range of locations and harvest years, many of the QTL detection methods ignore two- and three-way interactions between QTL, harvest, and location. In this work, a strategy for QTL detection in multi-harvest-location trial data, based on interval mapping and mixed models, is proposed and applied to map QTL effects on a segregating progeny from a biparental cross of pre-commercial Brazilian cultivars, evaluated at two locations and three consecutive harvest years for cane yield (tonnes per hectare), sugar yield (tonnes per hectare), fiber percent, and sucrose content. In the mixed model, we have included appropriate (co)variance structures for modeling heterogeneity and correlation of genetic effects and non-genetic residual effects. Forty-six QTLs were found: 13 QTLs for cane yield, 14 for sugar yield, 11 for fiber percent, and 8 for sucrose content. In addition, QTL by harvest, QTL by location, and QTL by harvest by location interaction effects were significant for all evaluated traits (30 QTLs showed some interaction, and 16 none). Our results contribute to a better understanding of the genetic architecture of complex traits related to biomass production and sucrose content in sugarcane.

A Simulation-Optimisation approach for hospital beds allocation.

The planning of hospital beds is among the most debated problems in healthcare. Despite being an important issue, many initiatives have failed to sustain services improvements, resulting in high costs and also high refusal rates. The stochastic problem involves conflicting criteria, therefore, we propose a Simulation-Optimisation approach to solve it. The Evolutionary Algorithm NSGA-II drives the process, and the solutions are validated and evaluated via Discrete Event Simulation. An application is performed in one of the health regions of the state of Minas Gerais, Brazil, where the public health system assists nearly 80% of the patients. The results pointed out that the proposed approach could find efficient and feasible solutions for the problem. Therefore, it is a good alternative to empirical methods currently used in Brazil to set hospital beds allocation.

Estimation of fetal weight by measurement of fetal thigh soft-tissue thickness in the late third trimester.

OBJECTIVE: The accuracy of current formulae for the sonographic estimation of fetal weight (EFW) is compromised by significant intra- and interobserver variability of biometrical measurements, particularly circumferences. The aim of this study was to assess the reliability of the linear measurement of mid-thigh soft-tissue thickness (STT) and to derive a novel formula for EFW. METHODS: This was a prospective study involving 388 singleton uncomplicated pregnancies. There were three consecutive phases: (1) to verify the relationship between STT and birth weight, (2) to derive a novel formula for EFW using femur length and STT only, and (3) to test the accuracy of the new equation. Only the 290 patients who delivered within 48 h of measurement were considered for the analysis. A comparison with other formulae was performed. RESULTS: STT was significantly correlated with both abdominal circumference and birth weight (r(2) = 0.36 and 0.46, respectively; P < 0.001). Both intra- and interobserver variability were satisfactory (0.44 +/- 0.27 and 0.57 +/- 0.35 mm, respectively). The equation for EFW was developed using multiple stepwise regression analysis (EFW = - 1687.47 + (54.1 x femur length) + (76.68 x STT)) and tested prospectively on 69 patients. The new formula yielded results (r = 0.79) that were slightly better in accuracy than two other published equations, and had an absolute mean error of < 15% in 97% of cases. CONCLUSIONS: Our findings confirm the potential of the linear measurement of mid-thigh STT as a valuable parameter for the sonographic assessment of fetal growth and EFW. Our new equation is apparently at least as reliable as the most widely used formulae for EFW.

The brain decade in debate: VII. Neurobiology of sleep and dreams.

This article is a transcription of an electronic symposium held on February 5, 2001 by the Brazilian Society of Neuroscience and Behavior (SBNeC) during which eight specialists involved in clinical and experimental research on sleep and dreaming exposed their personal experience and theoretical points of view concerning these highly polemic subjects. Unlike most other bodily functions, sleep and dreaming cannot, so far, be defined in terms of definitive functions that play an ascribable role in maintaining the organism as a whole. Such difficulties appear quite clearly all along the discussions. In this symposium, concepts on sleep function range from a protective behavior to an essential function for maturation of the nervous system. Kleitman's hypothesis [Journal of Nervous and Mental Disease (1974), 159: 293-294] was discussed, according to which the basal state is not the wakeful state but sleep, from which we awake to eat, to protect ourselves, to procreate, etc. Dreams, on the other hand, were widely discussed, being considered either as an important step in consolidation of learning or simply the conscious identification of functional patterns derived from the configuration of released or revoked memorized information.

Rapid eye movements during paradoxical sleep in patients with cerebrovascular disease.

Rapid eye movements that occur during paradoxical sleep are generated from the brainstem and are modulated by cerebral hemispheres. In an attempt to establish the participation of cerebral hemispheres on rapid eye movements, we carried out a quantitative study of eye movements density in patients bearing hemispheres vascular lesions. The polysomnographic recordings of 24 patients were compared to those of 24 healthy volunteers. Density of rapid eye movements was defined as the percentage of eye movements during the respective time of paradoxical sleep. Based on the present results, we concluded that: stroke patients with hemispheric lesions displayed increased density of rapid eye movements; there was no difference on the density of rapid eye movements according to the hemispheric lesion; higher density of rapid eye movements was observed in patients with anterior hemispheric lesion.

[Prognostic-evolutive evaluation of patients in coma based on ocular reflexes]. / Avaliação prognóstico-evolutiva dos doentes em coma mediante os reflexos oculares.

The ocular reflexes (ciliospinal, photomotor, oculocephalic, vestibulo-ocular, corneal) were studied in 84 comatose patients. The scope of the investigation was the assessment of each reflex in terms of its resistance according to the degree of depth of the coma. From the statistical viewpoint it was possible to emphasize the photomotor, corneal and vestibulo-ocular as the most resistant ones. The periodic evaluation in short time intervals of these five reflexes, observing their extinction or their re-appearance, permits a better prognostic assessment in a longitudinal viewpoint of the comatose patient.

Characterization of new polymorphic functional markers for sugarcane.

Expressed sequence tags (ESTs) offer the opportunity to exploit single, low-copy, conserved sequence motifs for the development of simple sequence repeats (SSRs). The authors have examined the Sugarcane Expressed Sequence Tag database for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 342 EST-SSRs, which represent a subset of over 2005 SSR-containing sequences that were located in the sugarcane EST database, could be designed from the nonredundant SSR-positive ESTs for possible use as potential genic markers. These EST-SSR markers were used to screen 18 sugarcane (Saccharum spp.) varieties. A high proportion (65.5%) of the above EST-SSRs, which gave amplified fragments of foreseen size, detected polymorphism. The number of alleles ranged from 2 to 24 with an average of 7.55 alleles per locus, while polymorphism information content values ranged from 0.16 to 0.94, with an average of 0.73. The ability of each set of EST-SSR markers to discriminate between varieties was generally higher than the polymorphism information content analysis. When tested for functionality, 82.1% of these 224 EST-SSRs were found to be functional, showing homology to known genes. As the EST-SSRs are within the expressed portion of the genome, they are likely to be associated to a particular gene of interest, improving their utility for genetic mapping; identification of quantitative trait loci, and comparative genomics studies of sugarcane. The development of new EST-SSR markers will have important implications for the genetic analysis and exploitation of the genetic resources of sugarcane and related species and will provide a more direct estimate of functional diversity.

Development of an integrated genetic map of a sugarcane (Saccharum spp.) commercial cross, based on a maximum-likelihood approach for estimation of linkage and linkage phases.

Sugarcane (Saccharum spp.) is a clonally propagated outcrossing polyploid crop of great importance in tropical agriculture. Up to now, all sugarcane genetic maps had been developed using either full-sib progenies derived from interspecific crosses or from selfing, both approaches not directly adopted in conventional breeding. We have developed a single integrated genetic map using a population derived from a cross between two pre-commercial cultivars ('SP80-180' x 'SP80-4966') using a novel approach based on the simultaneous maximum-likelihood estimation of linkage and linkage phases method specially designed for outcrossing species. From a total of 1,118 single-dose markers (RFLP, SSR and AFLP) identified, 39% derived from a testcross configuration between the parents segregating in a 1:1 fashion, while 61% segregated 3:1, representing heterozygous markers in both parents with the same genotypes. The markers segregating 3:1 were used to establish linkage between the testcross markers. The final map comprised of 357 linked markers, including 57 RFLPs, 64 SSRs and 236 AFLPs that were assigned to 131 co-segregation groups, considering a LOD score of 5, and a recombination fraction of 37.5 cM with map distances estimated by Kosambi function. The co-segregation groups represented a total map length of 2,602.4 cM, with a marker density of 7.3 cM. When the same data were analyzed using JoinMap software, only 217 linked markers were assigned to 98 co-segregation groups, spanning 1,340 cM, with a marker density of 6.2 cM. The maximum-likelihood approach reduced the number of unlinked markers to 761 (68.0%), compared to 901 (80.5%) using JoinMap. All the co-segregation groups obtained using JoinMap were present in the map constructed based on the maximum-likelihood method. Differences on the marker order within the co-segregation groups were observed between the two maps. Based on RFLP and SSR markers, 42 of the 131 co-segregation groups were assembled into 12 putative homology groups. Overall, the simultaneous maximum-likelihood estimation of linkage and linkage phases was more efficient than the method used by JoinMap to generate an integrated genetic map of sugarcane.

Randomized, double-blind clinical trial, controlled with placebo, of the toxicology of chronic melatonin treatment.

The objective of the present study was to assess the toxicology of melatonin (10 mg), administered for 28 days to 40 volunteers randomly assigned to groups receiving either melatonin (N = 30) or placebo (N = 10) in a double-blind fashion. The following measurements were performed: polysomnography (PSG), laboratory examinations, including complete blood count, urinalysis, sodium, potassium and calcium levels, total protein levels, albumin, blood glucose, triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), urea, creatinine, uric acid, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvate transaminase (GPT), bilirubin, alkaline phosphatase, gama-glutamic transaminase (GGT), T3, T4, TSH, LH/FSH, cortisol, and melatonin serum concentrations. In addition, the Epworth Somnolence Scale (ESS) and a sleep diary (SD) were also applied to the volunteers 1 wk before each PSG. In addition, the volunteers were asked about possible side effects (SE) that appeared during the treatment. The study was carried out according to the following timetable: Visit 0, filling out the term of consent and inclusion criteria; Visit 1, PSG, laboratory examinations, ESS, SD, melatonin serum concentrations; Visit 2, SD, melatonin serum concentrations, SE; Visit 3, melatonin serum concentrations, PSG, ESS, SE; Visit 4, laboratory examinations, SE, melatonin serum concentrations, SD; and Visit 5, PSG, ESS, SE. Analysis of the PSG showed a statistically significant reduction of stage 1 of sleep in the melatonin group. No other differences between the placebo and melatonin groups were obtained. In the present study we did not observe, according to the parameters analyzed, any toxicological effect that might compromise the use of melatonin at a dose of 10 mg for the period of time utilized in this study.

Juvenile fibromyalgia: clinical and polysomnographic aspects.

OBJECTIVE: To identify the child-mother diagnostic correlation in fibromyalgia (FM), to study sleep disturbance in juvenile FM, and to compare clinical aspects and sleep disorders between these groups. METHODS: We studied 34 children with confirmed FM aged 11 +/- 1 years, 10 children with diffuse pain, and 17 age and sex matched asymptomatic controls. The respective 61 mothers were included: 34 asymptomatic and 27 with FM. All participants were subjected to clinical evaluation, a sleep questionnaire, and nocturnal polysomnography, preceded by a night of adaptation. Sleep scoring was done visually and a computerized analysis was performed for alpha, theta, and delta waves in slow wave sleep (SWS). RESULTS: A significant predominance of mothers with FM was observed in the group of children with FM (71%) compared to children with diffuse pain (30%) and asymptomatic children (0%). According to the sleep questionnaire, the complaints of superficial sleep and nonrestorative sleep were more prominent in mothers with FM than in children with FM, whereas motor agitation during sleep was more frequent in the children with FM. Polysomnographic anomalies were also more prominent in mothers with FM than in children with FM in terms of decrease in sleep efficiency, increase of number of arousals during sleep, and alpha intrusion in SWS. Both FM groups presented an increased alpha + theta time/delta time index during SWS compared to respective controls, and mothers with FM also showed an increase in alpha time/delta time index during SWS, compared to asymptomatic mothers. A correlation was found between alpha + theta time/delta time index during SWS and intensity of clinical manifestations of pain and sleep anomalies in children and their mothers. CONCLUSION: Significant concordance was observed regarding FM diagnosis in children and their mothers. Sleep complaints and polysomnography findings were less prominent in affected children compared to mothers with FM. In addition, we observed a significant correlation between polysomnographic indexes, sleep anomalies, and pain manifestations in children and their mothers.