N-substituted benzimidazole acrylonitriles as in vitro tubulin polymerization inhibitors: Synthesis, biological activity and computational analysis.

We present the design, synthesis and biological activity of novel N-substituted benzimidazole based acrylonitriles as potential tubulin polymerization inhibitors. Their synthesis was achieved using classical linear organic and microwave assisted techniques, starting from aromatic aldehydes and N-substituted-2-cyanomethylbenzimidazoles. All newly prepared compounds were tested for their antiproliferative activity in vitro on eight human cancer cell lines and one reference non-cancerous assay. N,N-dimethylamino substituted acrylonitriles 30 and 41, bearing N-isobutyl and cyano substituents placed on the benzimidazole nuclei, showed strong and selective antiproliferative activity in the submicromolar range of inhibitory concentrations (IC50 0.2-0.6 µM), while being significantly less toxic than reference systems docetaxel and staurosporine, thus promoting them as lead compounds. Mechanism of action studies demonstrated that two most active compounds inhibited tubulin polymerization. Computational analysis confirmed the suitability of the employed benzimidazole-acrylonitrile skeleton for the binding within the colchicine binding site in tubulin, thus rationalizing the observed antitumor activities, and demonstrated that E-isomers are active substances. It also provided structural determinants affecting both the binding position and the matching affinities, identifying the attached NMe2 group as the most dominant in promoting the binding, which allows ligands to optimize favourable cation∙∙∙π and hydrogen bonding interactions with Lys352.

Expression of drought tolerance genes in tropical upland rice cultivars (Oryza sativa).

Gene expression related to drought response in the leaf tissues of two Brazilian upland cultivars, the drought-tolerant Douradão and the drought-sensitive Primavera, was analyzed. RNA-seq identified 27,618 transcripts in the Douradão cultivar, with 24,090 (87.2%) homologous to the rice database, and 27,221 transcripts in the Primavera cultivar, with 23,663 (86.9%) homologous to the rice database. Gene-expression analysis between control and water-deficient treatments revealed 493 and 1154 differentially expressed genes in Douradão and Primavera cultivars, respectively. Genes exclusively expressed under drought were identified for Douradão, including two genes of particular interest coding for the protein peroxidase precursor, which is involved in three distinct metabolic pathways. Comparisons between the two drought-exposed cultivars revealed 2314 genes were differentially expressed (978 upregulated, 1336 downregulated in Douradão). Six genes distributed across 4 different transcription factor families (bHLH, MYB, NAC, and WRKY) were identified, all of which were upregulated in Douradão compared to Primavera during drought. Most of the genes identified in Douradão activate metabolic pathways responsible for production of secondary metabolites and genes coding for enzymatically active signaling receptors. Quantitative PCR validation showed that most gene expression was in agreement with computational prediction of these transcripts. The transcripts identified here will define molecular markers for identification of Cis-acting elements to search for allelic variants of these genes through analysis of polymorphic SNPs in GenBank accessions of upland rice, aiming to develop cultivars with the best combination of these alleles, resulting in materials with high yield potential in the event of drought during the reproductive phase.

Discrimination of common bean cultivars using multiplexed microsatellite markers.

Analysis of DNA polymorphisms allows for the genetic identification and precise discrimination of species with a narrow genetic base such as common bean. The primary objectives of the present study were to molecularly characterize commercial common bean varieties developed at various research institutions using microsatellite markers and to determine the degree of genetic diversity among the bean varieties analyzed. Fifty cultivars representing 12 grain classes and 64 genitors, i.e., accessions used to develop these cultivars, were characterized. Based on an analysis of 24 simple sequence repeats, the estimates for the average number of alleles and genetic diversity were 8.29 and 0.646, respectively. The combined probability of identity was estimated at 7.05 x 10(-17), indicating a high individual discriminatory power. Thirty-two percent of the cultivars exhibited heterogeneity for multiple loci that reflected either homozygosity for different alleles of a given locus in different individuals or heterozygosity for the locus. The average genetic diversity for the groups of cultivars and genitors was 0.605 and 0.660, respectively, with no genetic differentiation (FST) between these groups. Although similar estimates of expected heterozygosity were observed when the cultivars were grouped by release date, a greater number of private alleles was observed in the most recent cultivars. The genetic differentiation among cultivars originating from different institutions was not different from zero (FST = 0.01). The molecular profile database derived from these analyses may increase the statistical power of genetic estimates and may be incorporated into breeding programs for common bean. Furthermore, the profiles obtained for the different cultivars may be used as molecular descriptors to complement traditional descriptors used in distinctiveness, uniformity and stability tests, thereby improving the traceability of samples and their derivatives and helping to protect the intellectual property rights of breeders.

Development and characterization of new microsatellites for Eugenia dysenterica DC (Myrtaceae).

Microsatellite markers were developed for population genetic analyses of the Neotropical tree Eugenia dysenterica DC (Myrtaceae), after construction of a shotgun genomic library for microsatellite discovery. Nine primers were designed, of which 5 yielded amplified product. These primers were polymorphic for 97 individuals collected in 3 distinct localities. The number of alleles per locus (primer) ranged from 3 to 11 and expected heterozygosities varied from 0.309 to 0.884. The probability of locus identity was ~1.88 x 10(-4) and the probability of paternity exclusion was ~0.9367. The 5 microsatellite primer pairs may be suitable for population genetic studies such as parentage and fine-scale genetic analyses of this species.

Molecular characterization of high performance inbred lines of Brazilian common beans.

The identification of germplasm genetic variability in breeding programs of the common bean (Phaseolus vulgaris) is essential for determining the potential of each combination of parent plants to obtain superior genotypes. The present study aimed to estimated the extent of genetic diversity in 172 lineages and cultivars of the common bean by integrating five tests of value for cultivation and use (VCU) that were conducted over the last eight years by the breeding program of Embrapa Arroz e Feijão in Brazil. Nine multilocus genotyping systems composed of 36 fluorescent microsatellite markers distributed across 11 different chromosomes of the common bean were used, of which 24 were polymorphic in all trials. One hundred and eighty-seven alleles were identified, with an average of 7.79 alleles per locus and an average gene diversity of 0.65. The combined probability of identity for all loci was 1.32 x 10(-16). Lineages that are more genetically divergent between the selection cycles were identified, allowing the breeding program to develop a crossbreed between elite genotypes with a low degree of genetic relatedness. HE values ranged from 0.31 to 0.63, with a large reduction in the genetic base over successive selection cycles. The test showed a significant degree of differentiation (FST = 0.159). Private alleles (26%) were identified and can be directly incorporated into the gene pool of cultivated germplasm, thereby contributing effectively to the expansion of genetic diversity in this bean-breeding program.

Working memory in Down syndrome: is there a dual task deficit?

BACKGROUND: Recent studies have shown that individuals with Down syndrome (DS) are poorer than controls in performing verbal and visuospatial dual tasks. The present study aims at better investigating the dual task deficit in working memory in individuals with DS. METHOD: Forty-five individuals with DS and 45 typically developing children matched for verbal mental age completed a series of verbal and visuospatial working memory tasks, involving conditions that either required the combination of two tasks in the same modality (verbal or visual) or of cross-modality pairs of tasks. RESULTS AND CONCLUSIONS: Two distinct deficits were found in individuals with DS: impairment in verbal tasks and further impairment in all dual task conditions. The results confirm the hypothesis of a central executive impairment in individuals with DS.

Development of microsatellite markers for the endangered Neotropical tree species Tibouchina papyrus (Melastomataceae).

We isolated and characterized 12 microsatellite loci for Tibouchina papyrus (Melastomataceae), an endangered species with narrow and disjunct range, endemics to a few localities in "cerrado rupestre" from Central Brazil. These microsatellites were obtained by sequencing of a genomic shotgun library for primer design. Leaves from 96 individuals collected in the three known local populations were genotyped using the 12 primers designed to analyze the polymorphisms at each locus. The number of alleles per locus ranged from one to six; two loci were monomorphic. Among the polymorphic loci, expected heterozygosities ranged from 0.161 to 0.714. Combined paternity exclusion probability was 0.957 and combined genetic identity (0.051) was high for studies on parentage. Tibouchina papyrus is a rare and endemic tree species of outcrop quartzite and sandstone soils, with highly isolated populations, which may have lead to the low degree of polymorphism that we detected. Also, motifs of most loci are larger than dinucleotide, which typically display lower levels of polymorphism.

Executive function in adolescents with Down Syndrome.

BACKGROUND: The present work is aimed at analysing executive function (EF) in adolescents with Down Syndrome (DS). So far, EF has been analysed mainly in adults with DS, showing a pattern of impairment. However, less is known about children and adolescents with this syndrome. Studying adolescents with DS might help us better understand whether performances on EF tasks of individuals with DS are determined by age or by Alzheimer disease, as some studies suggest, or whether their performances are directly related to DS cognitive profile. METHOD: A battery of EF tasks assessing set shifting, planning/problem-solving, working memory, inhibition/perseveration and fluency, as well as a tasks assessing sustained attention has been administered to a group of 15 adolescents with DS and 15 typically developing children matched for mental age. All EF tasks were selected from previous studies with individuals with intellectual disabilities or from developmental literature and are thought to be useful for the samples considered. RESULTS: The present results revealed that the group of individuals with DS performed at a significantly lower level on tasks assessing set shifting, planning/problem-solving, working memory and inhibition/perseveration, but not on the tasks assessing fluency. In addition, individuals with DS demonstrated a greater number of errors and less strategy use for the sustained attention task. CONCLUSIONS: The results suggest a broad impairment in EF in adolescents with DS, and are consistent with several similar studies conducted with adults with DS. We assume that EF deficit is a characteristic of DS.

Factors affecting false-negative rates on ex vivo sentinel lymph node mapping in colorectal cancer.

PURPOSE: Despite the increasing use of sentinel lymph node (SLN) mapping after colorectal cancer resection, reported node identification and false-negative rates vary considerably. The main aim of this prospective study was to quantify the false-negative rates on SLN mapping after resection and to evaluate factors influencing them. METHODS: Sixty-nine patients with biopsy-proven cancer of the colon and rectum underwent SLN mapping according to a protocol involving the ex vivo submucosal and peritumoral injection of 2-4 ml of Patent Blue V dye. All lymph nodes visualized were marked as SLN and totally embedded, then two 4 microm sections were cut for hematoxylin and eosin staining, and cytokeratin (AE1/AE3) immunostaining. A standard examination of the whole specimen and of the regional non-sentinel lymph nodes was also performed. RESULTS: SLNs were identified in 97.3% of the evaluable cases. A mean of 5.0 SLNs were removed per patient (SD+/-4.2). Nine false negatives were identified. Rectal cancer, tumor size>60mm, number of metastatic non-sentinel lymph nodes, and mucinous tumors were associated with false-negative SLNs. At multivariate analysis, a rectal location and mucinous differentiation were independently associated with false-negative SLNs. CONCLUSIONS: Ex vivo SLN mapping after colorectal cancer surgery is technically feasible with a high identification rate. Tumor size and stage, rectal involvement and a mucinous histology seem to interfere with the reliability of SLN staging. It is mandatory to standardize the procedure and selection criteria in order to deal with the question of the reliability of SLN mapping in colorectal cancer.

Rupture of the quadriceps tendon after arthroscopic lateral meniscectomy: A postoperative complication?

We report a case of complete quadriceps tendon rupture that occurred a few days after arthroscopic lateral meniscectomy. Complications following arthroscopy are rare; there have been many reports of quadriceps tendon rupture in the literature, but none by this kind of mechanism.

An unusual epidemic of Staphylococcus-negative infections involving anterior cruciate ligament reconstruction with salvage of the graft and function.

We performed a retrospective study of 13 patients who had postoperative clinical and laboratory signs of infection after autogenous bone-patellar tendon-bone (BPTB) anterior cruciate ligament (ACL) reconstructions. From January 1991 to November 1996 we experienced only 2 infected knees in 1,300 reconstructions, but between December 1996 and February 1997 10 patients in 70 ACL reconstructions developed a postoperative suspected infection. We found the origin of contamination (coagulase-negative Staphylococcus) in the supposedly sterile inflow cannula. When we changed this device, we had only 1 infection in the next 400 reconstructions. The diagnosis in these cases was derived from clinical signs and laboratory results, but only 2 of 11 samples of aspirated synovial fluid tested positive for Staphylococcus. The mean interval between the surgery and the onset of signs of infection and the start of antibiotic therapy was 7.7 days. All the patients had antibiotic association at the highest level. Six knees underwent arthroscopic debridement when the clinical signs indicated resistence to antibiotics. The normal postoperative rehabilitation program was modified but was not discontinued. Although recovery time was longer, overall results were similar to uncomplicated reconstructions. On the basis of our experience, we believe that when there is a notable increase in infection rates, a thorough search for contamination is indicated. Our source of infection was material that was thought to be sterile. Ultimately, early diagnosis and treatment is of critical importance to obtain good results. Even suspicion of infective postoperative complication should be sufficient cause to search for responsible microorganisms and begin antibiotic therapy. Arthroscopic debridement should be proposed to patients with resistence to antibiotics.

Three cases of patella fracture in 1,320 anterior cruciate ligament reconstructions with bone-patellar tendon-bone autograft.

Between September 1992 and December 1996 we reviewed three transverse displaced fractures of the patella occuring in 1,320 ACL reconstructions using bone-patellar tendon-bone autograft. All the patients suffered local injury to the donor knee between 8 and 12 weeks postoperatively. Immediate rigid fixation using single or double anterior tension band allowed early mobilization and full weight bearing. Between 6 and 9 months after fracture, the screws and the wire were removed and the grafts tested. Results of the pivot shift and Lachman test under anesthesia were negative and arthroscopic visualisation showed the graft to be intact. Postoperative assessment included the Lysholm and Tegner scales, the International Knee Documentation Committee Evaluation form (IKDC), KT-1000 arthrometer, and isokinetic dynamometer strength testing. No significant differences in the final outcome were noted between reconstructions complicated by patellar fracture and normal ACL reconstructions.

Intra-articular ACL reconstruction in the over-40-year-old patient.

We compared two groups of patients who had undergone anterior cruciate ligament (ACL) reconstruction. The groups were similar in regard to sex distribution, index injury, absence of meniscal or chondral lesions, surgical technique, and postoperative rehabilitation programs, but different in age. In the study group the mean age was 42.6 years, while in the control group the mean age was 20 years. The clinical results were assessed at a mean of 29 months after surgery using the Lysholm score, International Knee Documentation Committee form, Tegner activity level, and the KT-1000 arthrometer test. No significant differences were reported. Standard X-ray studies showed no variation between the two groups in the postoperative period. Intra-articular ACL reconstruction using a bone-patellar tendon-bone graft can be recommended in selected patients over 40 years of age as an alternative to conservative treatment.

Preoperative and postoperative evaluation of prostate-specific antigen in localized prostatic cancer treated by radical prostatectomy.

Preoperative prostate-specific antigen (PSA) values were determined in 73 patients with clinically localized prostatic cancer and candidates for a radical procedure. Correlation of preoperative PSA with a final pathological stage was attempted. Only in 44.8% of our 22 patients with organ-confined disease was the PSA value within the normal range; in 17.3% of cases PSA values were higher than 20 ng/ml. 18.2% of the patients with locally advanced disease showed normal PSA values, while 45.5% had concentrations above 20 ng/ml. In the case of lymph node involvement, PSA values were normal in 22.7% of the cases. Our data indicate that no strict relationship can be suggested between PSA and the final pathological stage and grading of the tumor in patients who underwent radical prostatectomy.

Signal averaging of pre- and post-extrasystolic beats in patients with ventricular arrhythmias.

To evaluate the effects of premature ventricular beats on the impulse conduction of adjacent sinus cycles, we compared the high amplification signal-averaged electrocardiogram parameters of the pre- and post-extrasystolic beats with those of the remaining sinus cycle. According to the duration of filtered QRS (fQRS), to the voltage of root mean square of the terminal 40 ms (RMS 40) and to the duration of low amplitude terminal components of the sinus cycles, ventricular late potentials were detected in nine out of 29 subjects. Patients with an abnormal signal-averaged electrocardiogram exhibited a longer fQRS (146 +/- 6 versus 116 +/- 2 ms), a reduced RMS40 voltage (18 +/- 2 versus 80 +/- 10 microV) and a prolonged duration of less than 40 microV components (42 +/- 4 versus 17 +/- 2 ms). Analysis of the pre-extrasystolic beats did not reveal any significant variation in the above parameters, showing a mean difference of 0.44 +/- 2.4 ms; 0.02 +/- 1.14 microV; 1 +/- 1.9 ms and of -1.45 +/- 1.02 ms; 3.5 +/- 8.6 microV; -0.7 +/- 0.84 ms respectively, for patients with and without ventricular late potentials. In addition, no significant variation was observed when the post-extrasystolic beats were considered. These results indicate that the sinus cycles adjacent to premature ventricular discharges do not present variations of signal-averaged electrocardiogram parameters that may suggest an influence of the ectopic beats on their intramyocardial impulse propagation.

Children's understanding of death according to parents and pediatricians.

Thirty pediatricians and 30 parents were interviewed on (a) how and when children develop concepts about death, (b) children's emotional responses to the thought of death, (c) the possibility of dialogue between adults and children about death, and (d) differences in healthy and seriously ill children's conceptualization of death. Pediatricians were no different than parents in their opinions. Moreover, in some cases, less educated parents and older doctors tended to hold opinions that were even farther from reality. Overall, the results suggest that children, healthy or sick, are left alone with the problem of death and are not helped either by their parents or by their doctors.