Chiral x achiral multidimensional liquid chromatography. Application to the enantioseparation of dintitrophenyl amino acids in honey samples and their fingerprint classification.

Most HPLC enantiomer separations are performed with columns packed with a chiral stationary phase (CSP) operated with an achiral mobile phase. The intrinsically limited chemical selectivity of most CSPs to the simultaneous resolution of several pairs of enantiomers means that complex mixtures of diverse pairs of enantiomers cannot be resolved in a single run due to peak overlapping. Moreover, some drawbacks remain when the analyte is present in very complex samples containing other achiral compounds which can co-elute with the enantiomer peaks. Multidimensional chromatography becomes an option to increase peak capacity and resolve these samples. The aim of this work was to study an online fully comprehensive 2D-LC mode utilizing a combination of a chiral column in the first dimension and an achiral column in the second dimension. The 2D-LC system was built with an active flow splitter pump in order to easily adjust the volume of sample transferred into the second dimension and to independently optimize the flow rate in the first dimension. The present LCxLC method was optimized for the separation of amino acids present in honey samples, taking into account key parameters that influence the bidimensional peak capacity (orthogonality, sampling frequency, etc.). The amino acids have been preconcentrated on a cation-exchange column followed by derivatization. Several amino acids present in different honey samples have been identified and the data generated has been analyzed by principal component analysis.

Gene Expression of Disease-related Genes in Alzheimer's Disease is Impaired by Tau Aggregation.

Neuronal hyperexcitability linked to an increase in glutamate signalling is a peculiar trait of the early stages of Alzheimer's disease (AD) and tauopathies, however, a progressive reduction in glutamate release follows in advanced stages. We recently reported that in the early phases of the neurodegenerative process, soluble, non-aggregated Tau accumulates in the nucleus and modulates the expression of disease-relevant genes directly involved in glutamatergic transmission, thus establishing a link between Tau instability and altered neurotransmission. Here we report that while the nuclear translocation of Tau in cultured cells is not impaired by its own aggregation, the nuclear amyloid inclusions of aggregated Tau abolish Tau-dependent increased expression of the glutamate transporter. Remarkably, we observed that in the prefrontal cortex (PFC) of AD patient brain, the glutamate transporter is upregulated at early stages and is downregulated at late stages. The Gene Set Enrichment Analysis indicates that the modulation of Tau-dependent gene expression along the disease progression can be extended to all protein pathways of the glutamatergic synapse. Together, this evidence links the altered glutamatergic function in the PFC during AD progression to the newly discovered function of nuclear Tau.

Benefícios da condroitinase abc associada a células-tronco mesenquimais na lesão espinhal aguda em ratos / Benefits of chondroitinase abc associated with mesenchymal stem cells in acute spinal cord injury in rats

Com o objetivo de estudar o efeito da condroitinase associada às células-tronco mesenquimais na lesão aguda da medula espinhal, utilizaram-se 50 ratos Lewis, distribuídos igualmente nos grupos: controle negativo (CN), tratamento com placebo (PLA), condroitinase (CDN), células-tronco mesenquimais (CTM) e condroitinase mais células-tronco mesenquimais (CDN+CTM). Todos os animais tiveram a medula espinhal exposta por laminectomia, e os grupos PLA, CDT, CTM e CDT+CTM sofreram também trauma medular compressivo. Após sete dias, procedeu-se à reexposição da medula espinhal, quando os grupos PLA e CTM receberam 4µL de líquido cefalorraquidiano artificial via intralesional, e os grupos CDT e CDT+CTM receberam o mesmo líquido contendo 2,2U de condroitinase. Após 14 dias da cirurgia inicial, todos os animais receberam 0,2mL de PBS via endovenosa, contudo, nos grupos CTM e CDT+CTM, esse líquido continha 1x106 CTM. Avaliou-se a capacidade motora até o 28o dia pós-trauma e, posteriormente, as medulas espinhais foram analisadas por RT-PCR, para quantificação da expressão gênica para BDNF, NT-3, VEGF, KDR e PECAM-1, e por imunoistoquímica, para detecção das células-tronco GFP injetadas (anti-GFP), quantificação dos neurônios (anti-NeuN) e da GFAP e vimentina, para avaliação da cicatriz glial. As análises estatísticas foram realizadas com o auxílio do Prism 5 for Windows, com o nível de significância de 5%. Não houve diferença entre os grupos quanto à capacidade motora. O grupo CDT+CTM apresentou maior imunoexpressão de neurônios viáveis do que o placebo. No CTM, houve maior expressão dos fatores neurotróficos BDNF e VEGF. E no CDT, houve menor imunoexpressão de vimentina. Concluiu-se que a associação CDT+CTM favorece a viabilidade neuronal após o trauma, que o tratamento com CTM promove aumento na expressão dos fatores tróficos BDNF e VEGF e que o tratamento com condroitinase é efetivo na redução da cicatriz glial.(AU)

The aim of this work was to study the effect of chondroitinase associated with mesenchymal stem cells in acute spinal cord injury. Therefore, 50 Lewis rats were distributed in the following groups: negative control (NC), treatment with placebo (PLA), chondroitinase (CDT), mesenchymal stem cells (MSC), and chondroitinase associated with mesenchymal stem cells (CDT + MSC). All animals had their spinal cord exposed by laminectomy, and the groups named PLA, CDT, MSC and CDT + MSC also suffered compressive spinal cord trauma. After seven days, the spinal cord was re-exposed, when the PLA and MSCs groups received 4uL of artificial cerebrospinal fluid through the lesion, and the CDT group and CDT + MSC received the same fluid containing 2,2U of chondroitinase. 14 days after the first surgery, all animals received 0.2ml of PBS intravenously; however, the MSC and CDT + MSC groups received the same liquid also containing 1x106 MSCs. The motor skills were evaluated up to 28 days post-injury and, subsequently, the spinal cords were analyzed by RT-PCR for BDNF, NT-3, VEGF, PECAM-1 and KDR gene expression quantification, immunohistochemistry to detect injected stem cells GFP (anti-GFP), to quantify neurons (anti-NeuN), GFAP and detect vimentin in order to evaluate the glial scar. Statistical analyzes were performed by Prism 5 for Windows using a 5% level of significance. There was no difference between groups with regarding motor capacity. The CDT + MSC group showed increased immunoreactivity of viable neurons than placebo. In MSC, there was a greater expression of neurotrophic factors BDNF and VEGF. Also, there was less vimentin immunostaining in group CDT. It was concluded that CDT + MSC association promotes neuronal viability after trauma, in which treatment with MSC promotes increased expression of BDNF and VEGF trophic factors, and also that treatment with chondroitinase is effective in reducing the glial scar.(AU)

Chemometric tools improving the determination of anti-inflammatory and antiepileptic drugs in river and wastewater by solid-phase microextraction and liquid chromatography diode array detection.

An analytical method for the simultaneous determination of seven non-steroidal anti-inflammatory drugs (naproxen, ketoprofen, diclofenac, piroxicam, indomethacin, sulindac and diflunisal) and the anticonvulsant carbamazepine in river and wastewater is reported. The method involves pre-concentration and clean-up by solid-phase microextraction using polydimethylsiloxane/divinylbenzene fibers, followed by liquid chromatography with diode array detection analysis. Owing to the fact that river water samples did not contain interferences and no sensitivity changes due to sample matrix were observed, external calibration was implemented. Standardization was also applied in order to carry out the prediction step by preparing only two diluted standards that were subjected to the pre-concentration step and a set of standards prepared in solvent. For the analysis of wastewater samples, in contrast, it was necessary to implement standard addition calibration in combination with the multivariate curve resolution-alternating least squares (MCR-ALS) algorithm, which allowed us to overcome matrix effect and exploit the second order advantage. Recoveries ranging from 72% to 125% for all pharmaceuticals proved the accuracy of the proposed method in river water samples. On the other hand, wastewater sample recoveries ranged from 83% to 140% for all pharmaceuticals, showing an acceptable performance - considering this sample contains no modeled interferences.

Spinal cord injury: some epidemiological data. A review of 233 cases.

Two hundred thirty-three consecutive cases of traumatic para- and tetraplegia admitted to a department of Neurosurgery in Naples within ten years were reviewed and categorized according to etiology, level of lesion and clinical features. The age of the patients at the time of injury varied from 7 to 74 years with a mean age of 37.1 +/- 17.1 (male mean age = 35.8 +/- 16.7; female mean age = 41.5 +/- 18.2). The male/female sex ratio was 4.8. Of these patients 66 were paraplegic and 167 were tetraplegic. The analysis of the various causes of the spinal cord injury showed that the occupational activities take the first place (32.2%); injuries in recreational activities and road accidents accounted for the majority of the remaining cases.