Optimal excitation and emission wavelengths to analyze amino acids and optimize neurotransmitters quantification using precolumn OPA-derivatization by HPLC.

We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation (λex) and emission (λem) spectrum wavelengths of OPA-amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λex-λem. The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λex-λem) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λex-λem wavelengths, in OPA-amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.

Recurrence of stroke amongst women of reproductive age: impact of and on subsequent pregnancies.

BACKGROUND AND PURPOSE: The risk of recurrence of stroke after pregnancy is poorly known. METHODS: This was an observational study of women younger than 45 years of age with transient ischaemic attack (TIA), cerebral infarction (CI), cerebral venous thrombosis (CVT) or intracerebral hemorrhage (ICH) treated in a stroke unit (January 1996-2011). The clinical data were prospectively collected in a database. Information on reproductive history after stroke was obtained using telephone surveys (2011). The variables were demographic data, vascular risk factors, stroke type, outcomes, medical advice concerning pregnancies after stroke, number of pregnancies after stroke, neurological assessment during pregnancy, antithrombotic treatments during pregnancy/puerperium, fertility treatments administered and information about hemorrhagic/ischaemic stroke recurrence. RESULTS: Overall, 102 women were included: 24 TIA, 64 CI (four large vessel disease, 14 cardioembolic, 12 small vessel disease, 17 undetermined etiology, 17 uncommon etiology), 12 CVT and two ICH. Mean age at the time of first stroke was 35 (±7.5) years. Median follow-up was 7.4 years (range 1-17). Thirty-two pregnancies occurred in 27 patients (previous diagnosis: four TIA, 17 CI, five CVT and one ICH). One woman became pregnant using in vitro fertilization. Only eight pregnancies were followed up by a neurologist. Of 26 pregnancies without previous history of ICH, 18 (62%) underwent preventive antithrombotic treatment. No recurrence of stroke was observed during pregnancy/puerperium. Of the women without pregnancies after the first cerebrovascular event, four CIs and three TIAs were observed. CONCLUSION: The recurrence of stroke after pregnancy is very low, which should be considered when counseling these patients.

RNA aptamer-mediated interference of HCV replication by targeting the CRE-5BSL3.2 domain.

The RNA genome of hepatitis C virus (HCV) contains multiple conserved structural RNA domains that play key roles in essential viral processes. A conserved structural component within the 3' end of the region coding for viral RNA-dependent RNA polymerase (NS5B) has been characterized as a functional cis-acting replication element (CRE). This study reports the ability of two RNA aptamers, P-58 and P-78, to interfere with HCV replication by targeting the essential 5BSL3.2 domain within this CRE. Structure-probing assays showed the binding of the aptamers to the CRE results in a structural reorganization of the apical portion of the 5BSL3.2 stem-loop domain. This interfered with the binding of the NS5B protein to the CRE and induced a significant reduction in HCV replication (≈50%) in an autonomous subgenomic HCV replication system. These results highlight the potential of this CRE as a target for the development of anti-HCV therapies and underscore the potential of antiviral agents based on RNA aptamer molecules.

Metabolic syndrome in calcium oxalate stones: Is it so important in this type of lithiasis?

INTRODUCTION AND OBJECTIVE: The association of the metabolic syndrome with lithogenesis has been described, especially in uric acid stones. The aim of the work was to analyze the role of the metabolic syndrome in oxalocalcic lithogenesis. MATERIALS AND METHODS: Metabolic evaluation of 151 patients including biochemical, hormonal and 24-urine urine parameters, as well as characteristics associated with metabolic syndrome. The relationship between the characteristics associated with the metabolic syndrome and those related to lithogenesis was evaluated using Spearman's correlation coefficient (SCC), Student's t test and Fisher's exact test. RESULTS: The average body mass index (BMI) was 25.9 (SD 3.7). The median age was 51 years (18.6-84.8) and 64.9% were men. There were no statistically significant differences between hypertension and estradiol, testosterone, triglycerides or cholesterol (P=.191, .969, .454, .345, respectively). Regarding glucose, the mean was 114.5 and 93.5mg/dl in patients with and without hypertension (P=.000). The levels of glucose, estradiol, testosterone or cholesterol did not vary with proteinuria (P=.518, P=.227, P=.095, P=.218, respectively). The mean triglycerides were 185.6 and 108.2mg/dl in patients with and without proteinuria (P=.001). Hypertension and proteinuria were not associated (P=.586). BMI correlated with serum and urinary uric acid and urinary creatinine. CONCLUSIONS: There are few associations between the characteristics of the metabolic syndrome and the anomalies related to lithogenesis. Metabolic syndrome does not seem to have a relevant role in the development of oxalocalcic stones.

Hepatitis C virus infection and the role of a pharmaceutical care program.

PURPOSE: The design, implementation, and assessment of a comprehensive pharmaceutical care program (CPCP) for hepatitis C virus (HCV)-infected patients treated with direct-acting antivirals (DAA) are described. SUMMARY: The advent of DAA regimens has caused the evolution of the role of hospital pharmacists, leading to the development of more specialized models of pharmaceutical care. Three clinical pharmacists were incorporated into the pharmacy department of a general tertiary teaching hospital in Madrid, Spain, with the aim of developing and implementing a CPCP for HCV-infected patients. Pharmacists were responsible for proposing standards and local guidelines to physicians, monitoring adherence to guidelines, managing drug interactions and adverse drug events (ADEs), providing patient education, and evaluating health outcomes and costs. Implementation steps included (1) estimation of the healthcare demand and pharmacy resources, (2) definition of the workflow of the CPCP, (3) definition of the treatment care plan, for which tools were developed to support pharmaceutical validation, detection, and management of ADEs and drug-drug interactions, and (4) program assessment in terms of safety and cost-effectiveness. The pharmacists' interventions performed, severity of errors intercepted, and patients' satisfaction with the CPCP were also assessed. This CPCP demonstrates that the involvement of the pharmacist throughout the care plan prevents harmful medication errors in this population (0.1 per patient) and prompts significant cost savings (€1.2 million for 1,930 treated patients). CONCLUSION: The implementation of a CPCP developed by hospital pharmacists for patients treated with DAA for HCV infection is an effective approach for preventing harmful medication errors and improving cost- effectiveness.

Faecal Transplantation, Pro- and Prebiotics in Parkinson's Disease; Hope or Hype?

Faecal microbiome transplantation (FMT) is an attractive technique, because the administration is relatively simple and in general has a mild adverse effect pattern. Moreover, FMT consists of a broad mixture, which could be beneficial, because at this moment it is not known what type of changes in the microbiome are needed. However, except from a few cases no clinical data in Parkinson's disease (PD) is available yet. There is some indication that FMT might be beneficial in severe constipated PD patients, but the clinical data to support this are very scarce. So, actually there are no good data in the public domain to support FMT at this moment in PD patients. FMT at this moment is a black box with too many unanswered questions, also with respect to safety concerns. Only the administration of species of Lactobacillus and Bifidobacterium over a time period of four to twelve weeks has repeatedly proven to be effective in treating constipation in PD. Also, no solid clinical data are available about the possible effects of probiotic treatment on motor symptoms or progression of PD. Therefore, also probiotic treatments in PD should wait until better clinical data become available, in order to select the right target populations and to have good estimates of the clinical effects to be expected.

Direct costs of a home parenteral nutrition programme.

BACKGROUND & AIMS: Home parenteral nutrition (HPN) is a lifesaving treatment for people with chronic intestinal failure and its cost has been reported to be very high. The purpose of the present paper was to study the direct healthcare and non-healthcare costs associated with the HPN programme managed by a tertiary hospital. METHODS: Observational, retrospective study of all adult patients on HPN from 11.1.2014 to 10.31.2015 treated at Gregorio Marañón University Hospital (Madrid, Spain). An economic evaluation was undertaken to calculate the direct healthcare (HPN provision, outpatient monitoring and management of complications) and non-healthcare costs (transportation process) of the HPN programme. The variables were collected from medical records, the dispensary and the hospital's financial services. The unit costs were taken from official price lists. RESULTS: Thirty-two patients met the inclusion criteria. Total direct healthcare and non-healthcare costs amounted to €13,363.53 per patient (€124.02 per patient per day). The direct healthcare costs accounted for 98.32% of overall costs, while the non-healthcare costs accounted for the remaining 1.68%. HPN provision accounted for the majority of the costs (74.25%), followed by management of complications (21.85%) and outpatient monitoring (2.23%). CONCLUSIONS: The direct healthcare costs accounted for the majority of HPN expenditure, specifically HPN provision was the category with the highest percentage.

[Medical students' knowledge of cardiovascular risk factors]. / Estudio sobre el conocimiento de factores de riesgo cardiovascular en estudiantes de medicina.

OBJECTIVES: To evaluate cardiovascular risk factor awareness among medical students at the University of Navarra. MATERIAL AND METHODS: A descriptive cross-sectional study including 334 students of the 1st, 4th and 6th year. They answered an anonymous structured questionnaire, and the results were analyzed using SPSS 11.0. RESULTS: Most 4th and 6th year students correctly identified the cardiovascular risk factors (CVRF). The 4th year students obtained better results on lipid values while the 6th year students performed better on body mass index and arterial hypertension. 97.8% of the sample considered the Mediterranean diet to be a protective factor. CONCLUSION: University of Navarra's medical students are well informed about CVRF and their knowledge increases along the degree course.

Influence of season, distance and mixed loads on the physical and carcass integrity of pigs transported to slaughter.

To determine the influences of season, distance and multiple-farm loads on pigs transported to slaughter under Spanish commercial conditions, 496 journeys, involving 90,366 pigs, were analysed. Mortality, liveweight loss, transport yield, killing out percentage, carcass yield after overnight chilling and percentage of condemned and trimmed carcasses were recorded. Season significantly affected killing out percentage and trimmed carcasses, with the lowest values found in autumn and summer, respectively. Distance significantly affected liveweight loss and transport yield. Loads composed of pigs from a number of farms had more deaths and carcass problems. It can be concluded that, under Spanish climatic conditions, if journey planning and handling at loading are adequately performed, season and distance do not impair the welfare of slaughter pigs. On the other hand, loading at a number of farms, alone, or interacting with distance, can compromise pig welfare.

[Bilateral axillary brachial plexus block guided by multiple nerve stimulation and ultrasound in a multiple trauma patient]. / Bloqueo axilar bilateral del plexo braquial guiado por neuroestimulación múltiple y ultrasonidos en una paciente con politraumatismo.

We present the case of a woman with multiple wounds and injuries after attempted suicide by jumping from a high place. She had multiple craniofacial injuries and fractures of both forearms requiring emergency osteosynthesis. The neurosurgeons requested that a level of consciousness be maintained for frequent assessment; therefore it was decided to provide a bilateral axillary brachial plexus block. The procedure was carried out with the aid of a nerve stimulator to locate a triple response in the left arm (radial, medial and musculocutaneous nerves) and with both ultrasound and double nerve stimulation in the right arm (medial and radial nerves). Surgery proceeded without adverse events. The location of nerves or nerve roots with both ultrasound and stimulators was highly useful in this patient in need of bilateral brachial plexus blockade. This combination, and ultrasound in particular, might be the technique of choice because it offers an image in real time and assessment of the least amount of anesthetic that seems to be needed for achieving a block.

Reaction time and rhythm of movement in Huntington's disease.

Huntington disease (HD) is characterized by several hyperkinesias though motor slowness is also another cardinal in this disease. In addition, self-paced timing movements are also disturbed in HD, which may also affect several rhythmic voluntary movements such as gait. Motor slowness can be measured with clinical scales such as the Unified Huntington's Disease Rating Scale (UHDRS) and timed tests, but also with the reaction time (RT) paradigm. We evaluated RT as a measure of motor slowness in 30 patients with genetically confirmed Huntington's disease and 24 control subjects. We also evaluated self-paced timing precision (SPTP) by applying a simple software program devised by our group. Clinical assessment was performed according to the UHDRS, including motor section, total functional capacity (TFC) and cognitive section (verbal fluency test, symbol digit, and Stroop test) The mean values obtained for RT and SPTP were statistically different in HD as compared with those from controls (p<0.0005). We observed a statistically significant correlation between RT and TFC scores (rs=-0.57, p<0.005 Spearman's correlation) and also between SPTP values and TFC scores (rs=-0.40, p<0.05 Spearman's correlation). In addition, RT and SPTP significantly correlated with cognitive scores (including digit symbol, verbal fluency and Stroop tests). Simple tests such as RT and SPTP provide an objective evaluation of motor impairment in HD yielding measures that correlate with clinical assessment and functional disability.

Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA.

The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini-R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.

Nitric oxide induces differentiation in the NB69 human catecholamine-rich cell line.

The nitric oxide (NO) donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), induced differentiation of human neuroblastoma NB69 cells to a dopamine phenotype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 microM SNAP in serum-free-defined medium for 24 h. SNAP treatment did not increase the number of necrotic or apoptotic cells. However, a decrease in the number of viable cells was observed at 750 microM SNAP. In addition, a decrease in (3)H-thymidine uptake was detected at the highest dose of SNAP. An increase in the antiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapoptotic Bax and Bcl-xS protein levels were also detected by Western blot analysis after SNAP treatment. At low doses (50-125 microM), SNAP induced an increase in catecholamine levels, (3)H-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250-750 microM). The TH protein content, analyzed by Western blot, remained unchanged in SNAP-treated cells throughout the range of doses studied, when compared with the control group. SNAP produced a dose-dependent decrease in the glutathione (GSH) content of the culture medium, without altering intracellular GSH. In addition, cGMP levels and nitrite concentration, measured in the supernatant of SNAP-treated cells, increased in a dose-dependent manner, as compared to control levels. The guanylate cyclase inhibitor lH-[1,2, 4]oxadiazolo[4,3a]quinoxaline-l-one (ODQ) did not revert the SNAP-induced effect on (3)H-dopamine uptake to control values. These results suggest that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the post-transcriptional level through a cGMP-independent mechanism.

In vivo effects of nipecotic acid on levels of extracellular GABA and taurine, and hippocampal excitability.

The effect of nipecotic acid on the extracellular levels of the putative hippocampal inhibitory transmitters, gamma-aminobutyric acid (GABA) and taurine, and also excitability in the dentate gyrus, were studied in anaesthetised rats using the dialytrode technique. The dentate gyrus was perfused by means of a dialytrode , using Krebs-Ringer bicarbonate or nipecotic acid solutions. Dentate field potentials, evoked by electrical stimulation of the perforant pathway, were recorded. Nipecotic acid drastically elevated the levels of extracellular GABA and taurine and the amplitude of population spikes in the dentate was concomitantly decreased. These results indicate that changes in extracellular levels of endogenous GABA and/or taurine influence the excitability of the hippocampus.

Effects of dihydrokainic acid on extracellular amino acids and neuronal excitability in the in vivo rat hippocampus.

The effect of inhibition of the high-affinity uptake of glutamate on the extracellular concentration of amino acids and on neuronal excitability was studied in vivo in the hippocampus of the rat. The dentate gyrus or CA1 field were perfused through a dialytrode with Krebs-Ringer-bicarbonate or dihydrokainic acid solutions. The spontaneous electrical activity and evoked field potentials were recorded concomitantly at dendritic or somatic levels. The results showed that with dihydrokainic acid: the extracellular concentrations of both glutamate and taurine were markedly increased in both areas of the hippocampus, the response of taurine being greater in CA1, while other amino acids were unaffected; in the dentate gyrus, the field excitatory postsynaptic potential was decreased while the population spikes were augmented, indicating an increased excitability of the neuronal population. In CA1, both the excitatory postsynaptic potential and spikes were reduced in amplitude. These results indicate that changes in the extracellular concentration of endogenous glutamate influences excitability of the tissue and that inhibition of the uptake processes for putative amino acid neurotransmitters increases the postsynaptic action of synaptically-released endogenous amino acids.

Low chloride-dependent release of taurine by a furosemide-sensitive process in the in vivo rat hippocampus.

Extracellular amino acid levels and field potentials evoked by perforant pathway stimuli were studied in vivo by means of a dialysis device, perfusing the rat dentate gyrus with low chloride solutions. When balanced with acetate, these perfusions enhanced the granule cell population spike amplitude. A specific extracellular taurine enhancement occurred whenever Cl- was replaced by acetate solution, reaching an increase of 20-fold over the basal taurine levels when 125 mM Cl- was replaced, whereas other amino acids remained unchanged. A considerable degree of Cl- replacement with iodide was needed, however, to obtain significant increases of extracellular taurine. Perfusions with bromide instead of Cl- did not cause any change in levels of extracellular amino acids including taurine. Furosemide, an inhibitor of Cl- transport, greatly reduced the taurine increase evoked by the low extracellular concentration of permeant anions. This drug also inhibited the taurine release induced by perfusion with 9 mM K+. These findings indicate that the extracellular increase of taurine, evoked by low permeant anion concentrations, may result from the taurine release through a furosemide-sensitive process.

In vitro and in vivo characterization of neural stem cells.

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.

Localization of gliosis in the corpus striatum of mice following cortical lesions.

Localized lesions of the medial and lateral frontal cortex were used to study gliosis, neurofilament content and changes in synaptic density in the mouse striatum. Relationships between the sites of cortical lesions and the localization of changes in different regions of the striatum were examined after 3 and 12 weeks. Independent of the location of frontal cortex lesions, glial fibrillary acidic protein (GFAP) immunoreactivity was increased throughout the entire striatum after 3 weeks. Twelve weeks after lesioning, increases in GFAP were confined to the dorsomedial (DM) striatum following medial cortical lesions, and to the dorsolateral (DL) striatum following lateral cortical lesions, suggesting persistent gliosis only in areas of striatal deafferentation. It appears, therefore, that the mechanisms which induce gliosis after short and long time periods are different.

Determination of HCV RNA concentration by direct quantitation of the products from a single RT-PCR.

A novel method for the estimation of HCV RNA levels in vivo was developed, based on competitive RT-PCR. The use of the Tth DNA polymerase and 5' 32P-labeled antisense primer respectively reduced cross-contamination and permitted the direct quantification of viral loads by the analysis of the radioactivity of PCR products derived from a clinical sample and a competitive deleted template, separated previously on a polyacrilamide gel. A HCV fragment (H) and a competitive (deltaH) RNA templates were synthesized for optimizing the method. The minimal starting RNA detectable by RT-PCR was 40 copies. RT-PCR performed with ratios deltaH/H ranging from 1/1 to 1/20 revealed different relative percentages of both H and deltaH products, changing from 90% of deltaH product when the ratio was 1/1 to 5%, when it was 1/20. Regression analysis was adjusted to a linear model and served to further estimate HCV RNA loads from clinical samples. HCV RNA quantitation was carried out in 19 patients. Higher viral loads were related to type 1b infection and persistence of HCV RNA after interferon therapy. This method is simple, reproducible and useful for rapid estimation of HCV RNA load in vivo.