DNA barcoding for molecular identification of Gynerium sagittatum(Poales: Poaceae): genetic diversity in savannah genotypes from Córdoba, Colombia / Código de barras de ADN para la identificación molecular de Gynerium sagittatum (Poales: Poaceae): diversidad genética de genotipos de las sabanas de Córdoba, Colombia

Introduction: The fiber of the Gynerium sagittatum Aubl. P. Beauv is raw material for the elaboration of several handcrafts, which are symbols of Colombian cultural identity. In the manufacture process, different genotypes are used according to the fiber quality and the type of craftsmanship, but it is believed that Gynerium is a complex species, and to date, there is no agreement on whether these genotypes belong to the same species or to different species. Objective: The aim of this study was to quickly and accurately identify wild cane plants using the nuclear ribosomal internal transcribed spacer (ITS1+ITS2), three chloroplast regions (matK, rbcL, ycf1), and their combinations. Methods: Different tests were used for discrimination: (1) inter and intraspecific distances, (2) Best Match (BM), Best Close Match (BCM), and tree-based method (3) Neighbor Joining (NJ) and (4) maximum likelihood and bayesian inference in molecular data. Results: The results showed that BM and BCM approaches revealed the low rate of correct species identification for ITS+matK (33.3 %) and ITS (28.6 %) loci, showing similarity among sequences. These results were further supported by tree-based analyses, where all individual regions and the different gene combinations had a zero discrimination rate. Conclusions: all genotypes belong to the same species of wild cane, therefore existing morphological differences can be related to phenotypic plasticity.

Introducción: La fibra de Gynerium sagittatum Aubl. P. Beauv, es materia prima esencial para la elaboración de varias artesanías, que son símbolos de la identidad cultural colombiana. En el proceso de fabricación, se utilizan diferentes genotipos de acuerdo con la calidad de la fibra y el tipo de artesanía, pero se cree que Gynerium es una especie compleja y hasta la fecha, no hay un consenso sobre si estos genotipos pertenecen a la misma especie o especies diferentes. Objetivo: Identificar de forma rápida y precisa plantas de caña silvestre utilizando el espaciador transcrito interno ribosomal nuclear (ITS1+ITS2), tres regiones de cloroplasto (matK, rbcL, ycf1) y sus combinaciones. Métodos: Se utilizaron diferentes pruebas para la discriminación: (1) distancias inter e intraespecíficas, (2) Prueba Best Match (BM), Best Close Match (BCM) y método basado en árboles (3) Neighbor Joining (NJ) y (4) Probabilidad de inferencia bayesiana mediante datos moleculares. Resultados: Los resultadosmostraron que los enfoques BM y BCM revelaron una baja tasa de identificación correcta de especies para los loci ITS+matK (33.3 %) e ITS (28.6 %), mostrando similitud entre las secuencias. Estos resultados fueron respaldados por análisis basados en árboles, donde todas las regiones individuales y las diferentes combinaciones de genes tuvieron una tasa de discriminación de cero (0 %). Conclusiones: los genotipos evaluados pertenecen a la misma especie de caña flecha y las diferencias morfológicas existentes pueden estar relacionadas con plasticidad fenotípica.

Floral micromorphology and nectar composition of the early evolutionary lineage Utricularia (subgenus Polypompholyx, Lentibulariaceae).

Utricularia (Lentibulariaceae) is a genus comprising around 240 species of herbaceous, carnivorous plants. Utricularia is usually viewed as an insect-pollinated genus, with the exception of a few bird-pollinated species. The bladderworts Utricularia multifida and U. tenella are interesting species because they represent an early evolutionary Utricularia branch and have some unusual morphological characters in their traps and calyx. Thus, our aims were to (i) determine whether the nectar sugar concentrations and composition in U. multifida and U. tenella are similar to those of other Utricularia species from the subgenera Polypompholyx and Utricularia, (ii) compare the nectary structure of U. multifida and U. tenella with those of other Utricularia species, and (iii) determine whether U. multifida and U. tenella use some of their floral trichomes as an alternative food reward for pollinators. We used light microscopy, histochemistry, and scanning and transmission electron microscopy to address those aims. The concentration and composition of nectar sugars were analysed using high-performance liquid chromatography. In all of the examined species, the floral nectary consisted of a spur bearing glandular trichomes. The spur produced and stored the nectar. We detected hexose-dominated (fructose + glucose) nectar in U. multifida and U. tenella as well as in U. violacea. In both U. multifida and U. tenella, there were trichomes that blocked the entrance into the throat and spur. Because these trichomes were rich in chromoplasts and contained lipid droplets, they may form an additional visual attractant. Bearing in mind the phylogenetic hypothesis for the genus, we suggest that an early ancestor of Utricularia had a nectariferous spur flower with a lower lip that formed a wide landing platform for bee pollinators.

Molecular phylogeny of bladderworts: A wide approach of Utricularia (Lentibulariaceae) species relationships based on six plastidial and nuclear DNA sequences.

The carnivorous plant genus Utricularia L. (bladderwort) comprises about 240 species distributed worldwide and is traditionally classified into two subgenera (Polypompholyx and Utricularia) and 35 sections, based mainly on general and trap morphology. It is one out of the largest carnivorous genera, representing ca. 30% of all carnivorous plant species, and is also the most widely distributed. According to previous phylogenetic studies, most infrageneric sections are monophyletic, but there are several incongruences considering their relationships and also the dissenting position of some species as a result of a too few (mostly one or two) molecular markers analyzed. Thus, here we present a multilocus phylogeny for Utricularia species with a wide taxonomic sampling (78 species and 115 accessions) based on six plastid (rbcL, matK, rpl20-rps12, rps16, trnL-F) and nuclear DNA (ITS region) sequences. The aim is to reconstruct a well-resolved tree to propose evolutionary and biogeographic hypotheses for the radiation of lineages with inferences about the divergence times of clades using a molecular clock approach.

[Confined placental mosaicisms a priori from meiotic origin: analysis of 10 cases]. / Anomalies chromosomiques limitées au placenta a priori d'origine méiotique : à propos de dix cas.

This retrospective monocenter study focused on confined placental mosaicisms a priori from meiotic origin (i.e. non-mosaic type 3 confined placental mosaicisms). From a series of 14,967 chorionic villus samplings performed in our Fetal Medicine Center, 10 non-mosaic type 3 confined placental mosaicisms were identified. These abnormalities only involved chromosomes 15, 16 or 22. Pregnancies complicated by these confined placental mosaicisms were associated with prematurity and neonatal hypotrophy. Thus, when a confined placental mosaicism is suspected, this retrospective study highlighted the need to characterize the type of confined placental mosaicism to prevent the probable intra-uterine growth retardation and to adapt the obstetrical monitoring if necessary.

The impact of early rehabilitation on the duration of hospitalization in patients after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: We examined the relationship between the improved physical activity by early rehabilitation and the duration of hospitalization among patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Thirteen allo-HSCT patients with myeloablative conditioning regimens (group A) and 13 patients with nonmyeloablative conditioning regimens (group B) were assessed retrospectively in this study. All patients received physical exercise immediately after neutrophil engraftment at the class 10,000 bioclean room (class 10,000). The mean daily steps at class 10,000 were measured as a substitute for the amount of physical activity, and the duration of hospitalization as one of the clinical outcomes. RESULTS: The degree of physical activity showed a negative correlation with the duration of hospitalization in group A (r = -.71; P = .0071), regardless of complications such as acute graft-versus-host disease, infections, and cytomegalovirus reactivation. However, there was no significant association in group B (r = .09; P = .77). CONCLUSION: The improved physical activity through early rehabilitation may be an independent, favorable prognostic factor for allo-HSCT patients with myeloablative conditioning regimens.

First-trimester prenatal diagnosis performed on pregnant women with fetal ultrasound abnormalities: the reliability of interphase fluorescence in situ hybridization (FISH) on mesenchymal core for the main aneuploidies.

OBJECTIVES: To examine the reliability of interphase FISH analysis of the main aneuploidies performed on mesenchymal core when prenatal diagnosis was performed on pregnant women with first-trimester fetal abnormalities on ultrasound. STUDY DESIGN: 386 first-trimester prenatal examinations were investigated from chorionic villus samplings for increased nuchal translucencies or other fetal ultrasound abnormalities. Interphase fluorescence in situ hybridization (FISH) for the main aneuploidies (trisomies 13, 18, 21 and gonosomal aneuploidies) was performed on the mesenchymal core of villi. Molecular cytogenetic results were always complemented by conventional cytogenetic results on long-term cultured villi (LTC-villi). Short-term cultured villi (STC-villi) preparations were retrospectively performed only when a chromosomal abnormality was observed with interphase FISH and/or LTC-villi. RESULTS: 88 chromosomal abnormalities (88/386=22.8% of first-trimester diagnoses) which could discuss subsequent abortions were observed after LTC-villi preparations. All cases possibly detectable by interphase FISH were detected. Thus, 85 aneuploidies (85/386=22.0% of first-trimester diagnoses; 85/88=96.6% of chromosomal abnormalities) were detected by interphase FISH, allowing early abortion by curettage before week 14 amenorrhea. No discrepancy occurred between interphase FISH and LTC-villi results for the aneuploidies studied. Three false-negative results (3/386=0.77% of first-trimester diagnoses; 3/88=3.41% of chromosomal abnormalities) were observed with STC-villi. CONCLUSION: We observed a high rate of false-negative results on cytotrophoblast cells. Conversely, interphase FISH of the main aneuploidies on the mesenchymal core provided rapid and reliable results, and therefore should be preferred to STC-villi in first-trimester prenatal diagnosis performed on pregnant women with fetal abnormalities on ultrasound.

Pain experience during chorionic villus sampling and amniocentesis: a preliminary study.

OBJECTIVE: To investigate the maternal perception of pain before and after amniocentesis (AC) or transabdominal chorionic villus sampling (TA-CVS). STUDY DESIGN: Three hundred women were divided into groups of 100 participants destined to undergo three different fetal sampling procedures: amniocentesis (group 1), transabdominal chorionic villus sampling (CVS) with a 19 gauge Blache needle (group 2) and transabdominal CVS with a 20 gauge needle (group 3). The visual analog scale (VAS) was used to quantify the patient's pre-sampling expected pain level and the real pain level was measured immediately after the sampling procedure. The factors liable to influence the VAS score after the sampling procedure were studied by single and multivariate analysis and concerned either the sampling procedure or patient demographic data. RESULTS: The VAS scores obtained before the procedure were not significantly different for the three sampling groups. When performed with a 19 gauge Blache needle TA-CVS is significantly more painful than the other sampling procedures (p=0.0002): VAS score of 3.62 (group 2), 2.49 (group 3) and 2.68 (group 1) for CVS with 20 gauge needle and amniocentesis. Multivariate analysis identified a group of patients for which the perception of pain induced by sampling was higher compared to the other patients: nulliparous patients, having undergone 19 gauge Blache needle CVS, with a high pre-sampling VAS score. CONCLUSION: Transabdominal chorionic villus sampling with a 19 gauge Blache needle seems to be the most painful sampling procedure. We question the need to use a 19 gauge needle as acceptable results are obtained with a 20 gauge needle.

Mosaic maternal uniparental isodisomy for chromosome 7q21-qter.

Uniparental disomy (UPD) for several human chromosomes is associated with clinical abnormalities. We report the case of a 2-year-old boy with severe intrauterine and post-natal growth retardation (IUGR/PNGR) and highly variable sweat chloride concentrations. The patient was identified as heterozygous for the F508del mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Unexpectedly, the signal corresponding to the maternally inherited F508del allele appeared much more intense than the paternally derived wild allele. Molecular analysis including polymorphic marker studies, microsatellites and single-nucleotide polymorphisms subsequently showed that the boy was a carrier of a de novo mosaic maternal isodisomy of a chromosome 7 segment while there was a biparental inheritance of the rest of the chromosome. This is the first report of a mosaic partial UPD7. The matUPD7 segment at 7q21-qter extends for 72.7 Mb. The karyotype (550 bands) of our patient was normal, and fluorescence in situ hybridization with probes mapping around the CFTR gene allowed us to rule out a partial duplication. The detection of this chromosomal rearrangement confirms the hypothesis that the 7q31-qter segment is a candidate for the localization of human imprinted genes involved in the control of IUGR and PNGR. It also emphasizes the importance of searching for UPD7 in severe, isolated and unexplained IUGR and PNGR.

Anchorage on fibronectin via VLA-5 (alpha5beta1 integrin) protects rheumatoid synovial cells from Fas-induced apoptosis.

BACKGROUND: Rheumatoid synovial cells are resistant to apoptosis induction in vivo, whereas, fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) are vulnerable to Fas-induced apoptosis in vitro. OBJECTIVE: To clarify this discrepancy by studying the contribution of the interaction between cellular integrin and matrix fibronectin (Fn), which is significantly increased in the rheumatoid joints, to the induction of apoptosis in RA-FLS. METHODS: Integrin and Fas mRNAs were measured by reverse transcription-polymerase chain reaction in RA-FLS. Integrins expressed in rheumatoid synovial tissues were analysed by immunohistochemistry. RA-FLS plated either on Fn or on control poly-L-lysine were incubated with agonistic anti-Fas monoclonal antibodies (mAbs). Apoptosis induction was evaluated using terminal deoxynucleotidyl transferase mediated UTP nick end labelling (TUNEL) and immunoblotting for caspase-3 and poly (ADP-ribose) polymerase in the presence or absence of anti-VLA-5 mAb. RESULTS: VLA-5 (alpha5beta1 integrin), a major integrin expressed on RA-FLS, was required for the adhesion of RA-FLS on Fn. RA-FLS plated on Fn were more resistant to Fas-induced apoptosis than those plated on control poly-L-lysine. This protection by Fn was reversed by anti-VLA-5 mAb. CONCLUSION: Anchorage of RA-FLS on matrix Fn via VLA-5 protects RA-FLS from Fas-induced apoptosis, and Fn abundantly present in rheumatoid synovium appears to afford RA-FLS resistance against apoptosis induction in vivo.

Differential expression of the nm23 genes in the developing human trophoblast.

NDP kinases are the non-specific enzymes which catalyse the synthesis of the NTPs through a transfer reaction using ATP as phosphoryl donor. In addition to their enzymatic activity, they display other not yet explained functions related to cell growth, differentiation and apoptosis, embryonic development, tumour progression and metastasis. In this study, the expression patterns of the three highly related NDP kinases A, B and C isoforms were investigated in the developing human trophoblast. Both NDP kinase A and B were found to be primarily present in the villous and extravillous cytotrophoblasts, while NDP kinase C was found almost exclusively in the syncytiotrophoblast layer. This suggests that NDP kinase A and B could be a marker for the mononuclear stage of differentiation of villous trophoblasts, while NDP kinase C could be a marker of the syncytiotrophoblast layer.

Pregnancy outcome and prognosis in fetuses with increased first-trimester nuchal translucency.

OBJECTIVE: One of the concerns of prenatal diagnosis is to find sensitive markers to screen for chromosome abnormalities, such as serum assays or nuchal translucency (NT). This study reports our experience with NT measurement during the first trimester of pregnancy. MATERIALS: The study was performed prospectively on 252 fetuses with either NT > or =3 mm or cystic hygroma. RESULTS: We observed 50 abnormal karyotypes, i.e. 19.8%. The incidence of chromosome abnormalities increased with increasing maternal age and increasing NT thickness. For the 202 fetuses with normal karyotypes, outcome was unfavourable in 32 cases: 23 elective terminations of pregnancy, 8 spontaneous abortions and 1 neonatal death. Outcome was favourable in 141 cases. Twenty-nine pregnancies were lost to follow-up. CONCLUSION: Measurement of NT at 12 weeks' gestation seems to be a good marker for chromosome abnormalities. When the karyotype is normal, the pregnancy outcome remains correlated with the degree of NT thickness. The finding of NT >3 mm between 10 and 14 weeks' gestation dictates rigorous ultrasound monitoring and caution when predicting pregnancy outcome.

The antiproliferative effect of mizoribine on rheumatoid synovial fibroblast mediated by induction of apoptosis.

In order to investigate the mechanism of anti-rheumatic action of mizoribine (MZR), antiproliferative effect of MZR on synovial fibroblasts obtained from rheumatoid arthritis (RA) patients were examined. To examine the effect of MZR on DNA synthesis, total radioactivity of 3H-thymidine (3H-TdR) incorporated into the synovial fibroblasts was measured. Also quantification of DNA fragmentation of synovial fibroblasts in the cultured supernatant and cell associated Bcl-2 protein, which is suspected of interfering with apoptosis, were performed with enzyme-linked immunosorbent assay. MZR suppressed 3H-TdR incorporation into synovial fibroblasts in a dose dependent fashion. Significant inhibition (P < 0.01) was attained at the concentration of more than 1 microgram/ml of MZR. However, induction of DNA fragmentation which is characteristic of apoptosis, were observed at only 10 micrograms/ml of MZR over 72 h-incubation significantly. In terms of the Bcl-2 expression of synovial fibroblasts, up to 10 micrograms/ml of MZR has no effect on the expression of this protooncogene bcl-2 expression. These results suggest that MZR might suppress the growth of rheumatoid pannus by inhibition of synovial fibroblast proliferation partially through the induction of apoptosis of synovial fibroblast without modulating Bcl-2 expression.

The role of cyclooxygenase-2 and inflammatory cytokines in pain induction of herniated lumbar intervertebral disc.

Lumbar disc herniation (LDH) is the disease which is the major cause of radiculopathy. In terms of the pathogenesis of disease, it is reported that prostaglandinE2 (PGE2) plays an important role to induce radiculopathy. Arachidonate cascade, which is the process of PGE2 synthesis, is mainly regulated by two kinds of enzymes, phospholipaseA2 (PLA2) and cyclooxy genase (COX). Previously, PLA2 was recognized as the rate-limiting enzyme of this cascade, and some authors reported the clinical significance of PLA2 at the site of LDH concerning the radicular pain. Recently, COX was elucidated to consist of 2 types of isoform, a constitutive form of COX-1 and an inducible form of COX-2. COX-2 has been focused as a key enzyme to regulate PGE2 synthesis and plays an important role in inflammation, because COX-2 was induced in many types of cells by the stimulation of inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). However, it is not fully discussed whether or not, COX-2 is induced in lumbar disc tissue and if it plays a significant role in the pathogenesis of LDH. To clarify the role of COX-2 in the pathomechanism of radiculopathy of LDH, we have investigated the expression of COX-2, IL-1 beta and TNF alpha in herniated lumbar disc tissue. Immunohistologically, they were detected in the cytosol of chondrocytes constituting the disc tissue. RT-PCR showed that herniated lumbar disc-derived cells expressed mRNA of COX-2, IL-1 beta and TNF alpha in the presence of inflammatory cytokines in vitro. The disc-derived cells also produced much PGE2 by stimulating of inflammatory cytokines at the same time and this PGE2 production was distinctly suppressed by a selective inhibitor of COX-2, 6-methoxy-2-naphtyl acetic acids (6MNA). These results suggest that COX-2 and inflammatory cytokines might play a causative role in the radiculopathy of LDH through upregulating PGE2 synthesis.

Effect of prostaglandin E2 on nitric oxide synthesis in articular chondrocytes.

Both nitric oxide (NO) and prostaglandin E2 (PGE2) are known to play an important role in cartilage metabolism. The present study investigated the novel intercellular mechanism of inducible NO synthase (iNOS) induction mediated by PGE2 in articular chondrocytes. Bovine articular chondrocytes were stimulated by cyclic adenosine monophosphate (cAMP) elevating agents like PGE2 in the presence of interleukin-1 alpha (IL-1 alpha). NO generation was measured by using the Griess reaction. Inducible NOS mRNA was semi-quantitated by reverse transcription-polymerase chain reaction (RT-PCR). While little NO was released from articular chondrocytes in the presence of PGE2 or direct adenylate cyclase activator such as forskolin, synergistic augmentation of NO generation was observed when chondrocytes were stimulated by PGE2 or forskolin in combination with IL-1 alpha. Further expression of iNOS mRNA by stimulation of PGE2 in the presence of IL-1 alpha simultaneously was also detected by RT-PCR in comparison with the mRNA induction by IL-1 alpha stimulation alone. These results indicated that PGE2 might modulate the articular cartilage metabolism by augmentation of chondrocyte NO synthesis in inflammatory process through cAMP-protein kinase A system.

Intervertebral disc cell apoptosis by nitric oxide: biological understanding of intervertebral disc degeneration.

While the unphysiological mechanical load is a central etiologic factor of disc degeneration, biologic factors including nitric oxide (NO) seems to play an important role in this condition. It is, therefore, investigated whether NO is related to the degeneration of intervertebral disc by way of inducing the disc cell apoptosis. Twelve herniated lumbar disc and eight control specimens were obtained from the patients underwent surgery. Apoptotic cells were identified by TUNEL procedure and the percent apoptotic cell index (ACI%) of each sample was calculated. Detection of iNOS expression was performed by immunohistochemical analysis. Disc cell monolayer culture was prepared from the surgical specimen of the patients with lumbar disc herniation. NO generation of the disc cells was measured by Griess reaction. Cell proliferation was detected by measuring the incorporation of 3H-Thymidine. The extent of fragmented DNA induced by NO donor, NOC-18, was also measured by an enzyme-linked immunosorbent assay. The incidence of apoptotic cell death (ACI%) was greater in the herniated group (61.3 +/- 24.5%) than that of control group (5.6 +/- 6.8%; P < 0.001). iNOS positive disc cells were detected in all the samples. NO production of disc cells was enhanced by the stimulation of IL-1 alpha. Suppression of 3H-Thymidine incorporation and DNA fragmentation in the disc cells were promoted by treatment of 100 microM NOC-18. These results suggest that disc cells are able to release NO and NO may play an important role in the pathogenesis of disc degeneration through the induction of apoptosis of disc cells in situ.

Induction of apoptosis in bovine articular chondrocyte by prostaglandin E(2) through cAMP-dependent pathway.

OBJECTIVE: Regulation of important biological processes such as proliferation and differentiation of articular chondrocytes is known to be mediated by prostaglandin E(2) (PGE(2)) in both normal and pathological states. Articular chondrocytes also undergo apoptosis, a biological phenomenon implicated in many physiological processes. Whether or not PGE(2) induces apoptosis in articular chondrocytes, however, is not known. DESIGN: Bovine articular chondrocytes were cultured with or without PGE(2) for 24 h and amounts of fragmented DNA, which is a distinct characteristic of apoptosis, were measured by enzyme-linked immunosorbent assay. Also effect of cyclic AMP (cAMP), which is one of the intracellular downstream mediator of PGE(2), on chondrocyte apoptosis was investigated. RESULTS: Administration of exogenous PGE(2) on bovine articular cartilage grown as a monolayer culture resulted in the induction of DNA fragmentation. This DNA fragmentation was accompanied with a marked dose-dependent increase in intracellular cAMP. Also cultured cells were treated with cAMP analogue, dibutyryl-cAMP or forskolin, a direct activator of adenylate cyclase, and the incidence of apoptosis in the chondrocytes was determined. As well as PGE(2), dibutyryl-cAMP and forskolin stimulated chondrocyte DNA fragmentation. CONCLUSIONS: It is the first report that PGE(2) can induce articular chondrocyte apoptosis in vitro. It is also suggest that apoptosis of chondrocytes by PGE(2) is linked with cAMP-dependent pathway.

[The long-term result of implant arthroplasty for hallux valgus deformity in rheumatoid arthritis].

We have reviewed the results of reconstruction of the forefoot in rheumatoid patients with arthroplasty using a Swanson flexible hinge toe implant for the great toe and with resection arthroplasty for the lateral four toes. The follow-up averaged 8 years (range, 5-11 years). All patients were female and an average age was 52 (range, 31-72 years). Preoperative and postoperative hallux valgus angle (HVA), the angles between the axes of the first and second metatarsal shafts (M1-M2) and the first and fifth metatarsal shafts (M1-M5) were measured radiographically. Breakage of the implant and radiolucencies around the implant on X-ray were evaluated. Preoperative average HVA, M1-M2 and M1-M5 were 31, 10, and 30 respectively. Postoperative average HVA, M1-M2, and M1-M5 were 17, 10, and 28 respectively. Breakage of the implant was present in 77% of the feet and the radiolucencies around the implant was present in 63% of the feet. Although only one foot was performed revision surgery for severe pain and breakage of the implant, more than 95% of the feet obtained pain relief. We found that this type of operation was very effective in relieving pain in rheumatoid patients.