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Anticholinergic burden tools have relevant pharmacological gaps that may explain their limited predictive ability for clinical outcomes. The aim of this study was to provide a universal pharmacological-based list of drugs with their documented affinity for muscarinic receptors. A comprehensive literature review was performed to identify the anticholinergic burden tools. Drugs included in these instruments were searched in four pharmacological databases, and the investigation was supplemented with PubMed. The evidence regarding the potential antagonism of the five muscarinic receptors of each drug was assessed. The proportion of drugs included in the tools with an affinity for muscarinic receptors was evaluated. A universal list of drugs with anticholinergic activity was developed based on their documented affinity for the different subtypes of muscarinic receptors and their ability to cross the blood-brain barrier. A total of 23 tools were identified, including 304 different drugs. Only 48.68%, 47.70%, 48.03%, 43.75%, and 42.76% of the drugs had an affinity to the M1, M2, M3, M4, and M5 receptor, respectively, reported in any pharmacological database. The proportion of drugs with confirmed antagonism varied among the tools (36.8% to 100%). A universal pharmacological-based list of 133 drugs is presented. It should be further validated in different clinical settings.

Summary Objective. To estimate the prevalence of self-reported adverse reactions (AdR) to subcutaneous airborne allergen immunotherapy (SCIT) and to describe factors associated with its occurrence. Methods. Real-life, observational, descriptive study of all patients treated with SCIT at a Portuguese allergy unit between 03/2017 and 06/2019, and who answered ≥1 time to a pre-SCIT evaluation questionnaire assessing the occurrence of local and/or systemic AdR in the previous administration. Results. 939 questionnaires from 231 patients (42% female, 35% with asthma) were included. Most (60%) SCIT preparations had multiple allergens with concentration adjusted to prevent dilution (MA-NoDil), 26% were single allergen with standard concentration (SA-SC), 10% single allergen with higher than standard concentration (SA-HC), and 4% mixtures without concentration adjustment (MA-Dil). SCIT-related AdR were self-reported in 313 (33%) administrations, 97% at the injection site and 11% grade 1 systemic symptoms. In a multivariable model, being a female and having asthma were associated with higher risk of AdR. MA-NoDil SCIT presented a lower risk of AdR compared to SA-SC SCIT. Conclusions. SCIT-related AdR were self-reported in 1/3 of the administrations, most at the injection site. The risk of AdR was higher in females and in patients with asthma. The lower risk of adverse reactions observed in SCIT preparations with multiple allergens with no dilutional effect should be further explored in future, targeted studies.

Fundamento: Medir o controlo da doença alérgica é fundamental na avaliação dos resultados da imunoterapia com alergénios (ITA). Objetivo: Descrever o controlo da doença alérgica (asma, rinite, conjuntivite) em doentes sob ITA subcutânea (ITSC). Métodos: Estudo descritivo, incluindo todos os indivíduos com >12 anos sob ITSC entre 03/2017 e 06/2019 nas unidades de Imunoalergologia da CUF‑Porto que responderam a ≥1 questionário de avaliação prévia à administração de ITSC em uso nas unidades; analisou -se apenas o questionário mais recente de cada indivíduo. O questionário incluiu uma escala visual analógica (EVA) de sintomas oculares (0, mínimo, a 10 cm, máximo) e o CARAT. O controlo da asma e rinite foi definido como CARAT total (CARATT)>24; o controlo das vias aéreas superiores (VAS) e inferiores (VAI) baseou -se nas pontuações parciais (CARATVAS>8 e CARATVAI≥16, respetivamente). Resultados: Incluíram -se 209 indivíduos, 96 (46%) do sexo feminino e 62 (30%) com

The physiological age of mother tubers (Solanum tuberosum L. cv. Kennebec) used as a source of material influenced kinetin-induced in vitro tuberisation. Tuberisation significantly increased with physiological age. Kinetin- or ancymidol-induced tuberisation, plantlet and microtuber dry weight decreased with increasing number of subcultures. Single-node segments obtained from tubers stored for more than 9.5 months at 4 °C showed increased kinetin-induced tuberisation rates and earlier tuberisation than those obtained from younger tubers. For any physiological age, material may be safely multiplied using node propagation until the third subculture and bioassayed for tuberisation without variation in the response.

Aims Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in cardiac remodelling. Available information regarding their prognostic utility in heart failure (HF) and cardiac resynchronization therapy (CRT) is controversial. The aim of this study was to analyse MMP-2 and TIMP-1 levels as predictors of long-term mortality in HF patients treated with CRT. Methods and results We prospectively included 42 consecutive patients with successfully implanted CRT. Matrix metalloproteinase-2 and TIMP-1 assays were performed prior to implant. Patients were evaluated at baseline and at the outpatient clinic at 6-month intervals. Clinical response, left ventricular (LV) remodelling, and mortality were analysed. During a mean follow-up of 60 ± 34 months, long-term mortality from any cause was 36% (15 patients). The cause of death was end stage of HF in 12 patients, sudden death in 2 patients, and 1 unknown. After adjustment using a Cox regression model, the independent predictors of long-term mortality were baseline TIMP-1, hazard ratio (HR) 1.18 (95% confidence interval (95% CI) [1.05–1.33], P = 0.007), baseline glomerular filtration rate (GFR), HR 0.97 (95% CI [0.94–1.00], P = 0.05), and permanent atrial fibrillation (AF), HR 3.14 (95% CI [1.02–9.67], P = 0.04). Area under receiver operating characteristic curve for TIMP-1 was 0.79 (95% CI [0.63–0.94]). Tissue inhibitor of matrix metalloproteinase-1 ≥ 248 ng/mL predicts mortality with 80% sensitivity and 71% specificity. Conclusion Tissue inhibitor of matrix metalloproteinase-1 is a powerful predictor of long-term mortality in HF patients treated with CRT.

Background— Pulmonary hypertension (PH) and collagen metabolism abnormalities are prevalent in patients with heart failure with preserved ejection fraction (HFpEF). Peripheral endothelial dysfunction (PED) has been described in HF and in pulmonary arterial hypertension. Our aim is to determine whether PH is associated with PED and impaired collagen metabolism in patients with HFpEF.; Methods and Results— Flow-mediated dilation of the brachial artery, matrix metalloproteinase-2 and matrix metalloproteinase-9, tissue metalloproteinase inhibitor 1, and C-terminal propeptide of type I procollagen were determined in 28 patients with HFpEF and 42 hypertensive controls. Patients with systolic pulmonary artery pressure >35 mm Hg on echocardiogram underwent a right heart catheterization. Patients with HFpEF had more severe PED than controls: flow-mediated dilation 1.95% (−0.81 to 4.92) versus 5.02% (3.90 to 10.12), P =0.002. Twenty patients with PH underwent right heart catheterization: mean pulmonary artery pressure 38 (27–52) mm Hg, wedge capillary pressure 18 (16–22) mm Hg, pulmonary vascular resistance 362 (235–603) dyn s cm -5 . There was a significant inverse correlation between flow-mediated dilation and pulmonary vascular resistance in patients with HFpEF and PH ( r =−0.679; P =0.002). Patients with HFpEF showed higher matrix metalloproteinase-2 and C-terminal propeptide of type I procollagen values than hypertensive controls. Patients with HFpEF and higher C-terminal propeptide of type I procollagen values also had higher mean pulmonary artery pressure ( r =0.553; P =0.014), transpulmonary gradient ( r =0.560; P =0.013), and pulmonary vascular resistance ( r =0.626; P =0.004). Conclusions— In patients with HFpEF, there is a significant correlation between PED and pulmonary vascular resistance. Collagen metabolism was more impaired in patients with HFpEF and PH. PED and collagen metabolism assessment could be useful tools to identify patients with HFpEF at risk of developing PH.

Aims A maximum percentage of ventricular pacing is mandatory to obtain a good response to CRT. Atrioventricular junction (AVJ) ablation has been recommended to attain this objective in patients with AF. The aims of our study were: (i) to determine whether the presence of complete AVJ block (induced or spontaneous) improves survival in patients with permanent AF treated with CRT and (ii) to analyse the predictors of mortality in AF patients treated with CRT. Methods and results From a series of 608 patients treated with CRT in our centre from 2000 to 2011, a cohort of 155 patients with permanent AF was analysed. Patients in AF were divided into two groups, AF + AVJ block [76 (49%)] and AF non-AVJ block [79 (51%)]. Mean follow-up was 30 months (interquartile range 13–51 months). During the follow-up, 62 patients died. Overall and cardiovascular mortality were similar between both groups: hazard ratio (HR) 0.85, 95% confidence interval (CI) 0.51–1.39, P = 0.51 and HR 0.94, 95% CI 0.52–1.68, P = 0.82. Multivariate analysis identified three independent predictors of mortality: basal NYHA functional class IV (HR 2.25, 95% CI 1.12–4.22, P = 0.03), glomerular filtration rate (HR 0.98, 95% CI 0.96–0.99, P = 0.03), and LVEF (HR 0.94, 95% CI 0.89–0.99, P = 0.02). Conclusions AVJ block did not improve survival for patients in AF treated with CRT. Basal NYHA functional class IV, poor renal function, and LVEF were the independent predictors of mortality.

Mutations that modify the amino acid sequence of C1-INH (except Val458Met) are associated with HAE. More than 200 different mutations scattering the entire C1-INH gene have been reported. The main objective of this study was to report the mutational findings in a HAE cohort of 138 Portuguese patients followed in specialized consultation all over the country. DNA was extracted from peripheral blood with QiaSymphony BioRobot (QIAGEN Portugal). The sequence reactions were performed by using a DNA sequencing kit (Big Dye terminator cycle sequencing v1.1/v3.1 from Applied Biosystems) and sequencing products were immediately submitted to direct sequencing on an Applied Biosystem 3130 DNA Analyser. DNA sequences were analyzed at four different stages. Raw data and sequence alignments of all 8 exons and intron–exon boundaries were performed for each patient individually with SeqScape software and using SERPING1 gene NG_009625 of 24,300 bp (12-March-2011) as reference sequence. Sequence comparisons among patients and controls were performed with software CodonCode Aligner v.3.7 from CodonCode Corp and with Geneious 4.5 from Biomatters Lda. A total of 94 point mutations were observed among patients, and 67% of them were located on exon 8. In addition, we noticed one not described stop codon at position c.1459 C>T in three different patients. Translation termination was also found on exon 3 and 7, as a result of mutations at positions c.481A>7, c.1174C>T. In this population, the prevalence of the missense mutation p.Arg444Cys was 39 out of 42. Mutational analysis revealed 22 different pathogenic mutations, of which 64% were not described on HAE database. Although identification of disease causing mutations is not necessary to establish HAE diagnosis, studies on gene expression and characterization of rearrangements in SERPING1 gene are suggested in order to get new insights on function and genetic tests of C1 inhibitor.

Indomethacin (IM), a non-steroidal anti-inflammatory drug, has the capacity to induce hepatic and renal injuries when administrated systemically. The aim of this study is to assess the IM absorption from complexed forms when orally administered to rats, by means of a comparative evaluation of its capacity to induce hepatic and renal injury in different forms, namely IM acid, IM sodium salt or IM complexed with hydroxypropyl-β-cyclodextrin (HP-β-CD), using freeze- and spray-drying methods. A total of 135 Wistar rats weighing 224.4 ± 62.5 g were put into 10 groups. They were allowed free access to water but were maintained on fast for 18 h before the first administration until the end of the experiment. Water and HP-β-CD (control groups) and IM acid form, IM trihydrated-sodium-salt and IM-HP-β-CD spray- and freeze-dried, at normal and toxic doses (test groups), were orally administered once/day for 3 days. Seventy-two hours after the first administration, the animals were sacrificed and a fragment of the liver and one kidney were collected and prepared for histopathological evaluation. Lesion indexes (rated 0/4 for liver and 0/3 for kidney) were developed and the type of injury scored according to the severity of damage. A statistical analysis of the severity and incidence of lesions was carried out. Animals administered with IM complexed forms showed similar hepatic and renal lesions, both in toxic and therapeutic doses, when compared with those observed in animals administered with IM acid or salt forms. This suggests that under the present experimental conditions, IM is equally absorbed from the gastrointestinal tract, independently of the administered IM form.

Aims Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a role in left ventricular structural remodelling. The aim of our study was to analyse MMP-2 and TIMP-1 levels as predictors of poor response to cardiac resynchronization therapy (CRT). Methods and results A cohort of 42 CRT patients from our centre was prospectively evaluated at baseline and after 12-month follow-up. MMP-2 and TIMP-1 assays were performed prior to CRT implant. Cardiac resynchronization therapy responders were defined as patients who survived, were not transplanted, and increased their basal 6 min walking distance test (6MWDT) by ≥10% or improved their NYHA functional class. Overall, 25 patients (60%) were classed as responders. At 12-month follow-up, six patients (14.2%) had died and one (2.4%) patient had been transplanted. Compared with responders, non-responders had higher levels of TIMP-1 (277 ± 59 vs. 216 ± 46 ng/mL, P = 0.001), MMP-2 (325 ± 115 vs. 258 ± 56 ng/mL, P = 0.02), and creatinine (1.76 ± 0.8 vs. 1.25 ± 0.3 mg/dL, P = 0.01). In a multivariate analysis, TIMP-1 was the only independent predictor of non-response to CRT [OR 0.97, 95% (CI 0.96–0.99) P = 0.005]. TIMP-1≥248 ng/mL predicted non-response with 71% sensitivity and 72% specificity. Conclusion TIMP-1 is an independent predictor of non-response in patients treated with CRT.

Previous research suggests 20% of omnivores and 40% of vegetarians do not consume enough vitamin B12. The contribution of seafood to dietary B12 intake stratified by frequency of seafood consumption and B12 status of young adults was assessed. Seafood was the greatest contributor to B12 intake (31%) among nonvegetarians, and intake comparisons based on frequency of seafood consumption revealed that frequent seafood consumers had higher (p < 0.001) B12 intakes. None of the frequent seafood consumers had suboptimal status, which occurred mainly in subjects consuming seafood < 1 time/month and vegetarians. Our findings suggest that modest seafood intake contributes to maintaining normal B12 status.

Indomethacin (IM) is a non-steroidal anti-inflammatory drug which inhibits prostaglandin biosynthesis. It is practically insoluble in water and has the capacity to induce gastric injury. Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is an alkylated derivative of beta-CD with the capacity to form inclusion complexes with suitable molecules. IM is considered to form partial inclusion complexes with HP-beta-CD by enclosure of the p-chlorobenzoic part of the molecule in the cyclodextrin channel, reducing the adverse effects. The aim of this paper is to evaluate the gastric damage induced by the IM inclusion complex prepared by freeze-drying and spray-drying. A total of 135 Wistar rats weighing 224.4 +/- 62.5 g were put into 10 groups. They were allowed free access to water but were maintained fasted for 18 h before the first administration until the end of the experiment. IM acid-form, IM trihydrated-sodium-salt and IM-HP-beta-CD spray and freeze-dried, at normal and toxic doses, were administered through gastric cannula once/day for 3 days. Seventy-two hours after the first administration, the animals were sacrificed and the stomachs collected and prepared for morphological study by using the haematoxylin-eosin technique. Lesion indexes (rated 0/4) were developed and the type of injury was scored according to the severity of damage and the incidence of microscopic evidence of harm. Microscopic assessment demonstrated levels of injury with index one on 10-25%. The type of complexation method had different incidence but the same degree. The results show that IM inclusion complexation protects against gastric injury, reducing the incidence and the maximum degree of severity from 4 to 1, with a better performance of the spray-dried complex.

Background: The traditional Mediterranean diet is claimed to possess antioxidant and immune-regulatory properties in several chronic diseases. Typical Mediterranean foods have recently been associated with improvement of symptoms of asthma and rhinitis in children. However the effect of adherence to Mediterranean diet on adult asthma outcomes is unknown. We aimed to investigate the association between adherence to Mediterranean diet and asthma control. Methods: Cross sectional study of 174 asthmatics, mean (SD) age of 40 (15) years. The patients were defined as controlled, in contrast to noncontrolled, if they showed FEV1 ‡ 80% of predicted, exhaled nitric oxide (NO) £ 35 ppb, and Asthma Control Questionnaire score

Histidine-tagged human cardiotrophin-1 (hCT-1), a recently discovered cytokine with excellent therapeutic potential, was expressed in tobacco chloroplasts under the transcriptional and translational control of two different promoters (rrn and psbA) and 5'-untranslated regions (5'-UTRs) (psbA and phage T7 gene 10). The psbA 5'-UTR promotes recombinant hCT-1 (rhCT-1) accumulation in chloroplasts at higher levels (eight-fold) than those obtained for the phage T7 gene 10 5'-UTR, regardless of the promoter used, indicating that the correct choice of translational control element is most important for protein production in chloroplasts. The maximum level of rhCT-1 achieved was 1.14 mg/g fresh weight (equivalent to 5% of total soluble protein) with the psbA promoter and 5'-UTR in young leaves harvested after 32 h of continuous light, although the bioactivity was significantly lower (approximately 35%) than that of commercial hCT-1. However, harvesting in the dark or after 12 h of light did not result in a significant decrease in the bioactivity of rhCT-1, suggesting that 32 h of over-lighting affects the biological activity of rhCT-1. Because high levels of rhCT-1 accumulation took place mainly in young leaves, it is proposed that seedlings should be used in a 'closed system' unit, yielding up to 3.2 kg per year of rhCT-1. This amount would be sufficient to meet the estimated annual worldwide needs of hCT-1 for liver transplantation surgery in a cost-effective manner. Furthermore, our strategy is an environmentally friendly method for the production of plant-based biopharmaceuticals.

Stored electrograms or marker channels are available in most of modern cardiac pacemaker models. We sought to analyse these information to uncover terminal events of pacemaker patients dying suddenly. Method and results We made post-mortem pacemaker (PM) interrogations in 19 patients dying suddenly out of hospital between the years 1997 and 2005 (mean age 59 +/- 13 years, 90% males). The systems had activated arrhythmia monitoring algorithms. Indications of pacing were sick sinus syndrome in seven, AV-block in five, and heart failure due to asynchrony in seven cases. The interrogated pacemakers were CHORUS 7034 (n = 12), CONTAK TR (n = 2), and INSYNC III (n = 5). For interpretation stored marker channels and electrograms were analysed. The mean observation time after PM implantation prior death was 2.11 +/- 1.44 years, the mean left ventricular ejection fraction from the last available echo examination in the year prior death was 27.5 +/- 8%, mean age was 63 +/- 12 years. In 17/19 cases (89%), a tachycardia (most likely ventricular tachycardia) was found correlating to the time of death. The mean cycle length of the terminal arrhythmia was 307 +/- 144 (250-344) ms, corresponding to a heart rate of 195 +/- 95 (174-240) bpm. We found no evidence of specific pacemaker-related problems such as electronic failure, battery depletion, or undersensing.Post-mortem analysis of arrhythmia monitoring of pacemaker patients revealed tachycardias (most likely ventricular tachycardia) to be related to sudden death. These findings give some insight in mechanisms of terminal events in this group.

Aims Coronary sinus (CS) lead implantation is a technically challenging procedure owing to variable vein anatomies and a high dislocation rate. Therefore, CS lead technology has undergone evolutionary changes during the last 10 years. The mode of fixation has been a passive one up to now. We want to describe our first clinical experience with the newly available active fixation lead 4195 in terms of dislocation rate and stability of thresholds compared with conventional models. Methods and results From 1999 to February 2007, we implanted 403 CS leads in 368 patients. Leads were categorized into three different groups on the basis of their fixation mechanism: straight (Easytrak I and Situs OTW; n ¼ 54), curved (Attain 4193 and 4194, Corox, Aescula, Situs ULD; n ¼ 308), and active (Attain 4195; n ¼ 41). Operative and follow-up data were prospectively noted and checked for significance between groups during the first 3 months after implantation. Kaplan–Meier analysis of long-term lead function was also performed. Straight and curved CS leads suffered from significantly more dislocations compared with active fixation (P , 0.001). The active fixation lead (4195) has a stable threshold over time compared with a significant rise after 24 h and thereafter in straight (62%) and curved leads (20%). However, retraction of an active fixation CS lead may be a difficult issue as outlined in two cases requiring pullback of a 4195 lead owing to phrenic nerve stimulation (one unsuccessful despite vigorous traction). Conclusion The active fixation lead 4195 using retention lobes yielded stable thresholds over time and seems to be superior to conventional leads in terms of dislocation. However, extraction may be a difficult or even impossible task.

Background: It has been hypothesized that the response of holo-transcobalamin (holo-TC) to oral vitamin B -12 may be used to assess absorption. To develop a reliable clinical absorption test that uses holo-TC, it is necessary to determine the optimal timeline for vitamin B-12 administration and postdose assessment. Objective: The objective of this study was to assess the magnitude and patterns of change in the postabsorption response of holo-TC to oral vitamin B-12. Design: Adult (18 -49 y) male and female participants (n = 21) with normal vitamin B-12 status were given three 9-μg doses of vitamin B-12 at 6-h intervals beginning early morning (baseline) on day 1. Blood was drawn at 17 timed intervals over the course of 3 d for the analysis of holo-TC and other indicators of vitamin B-12 status. Results: Mean holo-TC increased significantly (P < 0.001) from baseline at 6 h (11%) and 24 h (50%). TC saturation increased significantly (P < 0.001) from baseline at 12.5 h (33%) and 24 h (50%). The mean cobalamin concentration changed significantly (P

Treatment of gastric ulcers with medicinal plants is quite common in traditional medicine worldwide. Cymbopogon citratus (DC) Stapf. leaves infusion has been used in folk medicine of many tropical and subtropical regions to treat gastric disturbances. The aim of this study was to assess the potential gastroprotective activity of an essential oil-free infusion from C. citratus leaves in acute gastric lesions induced by ethanol in rat.The study was performed on adult male Wistar rats (234.0±22.7g) fasted for 24h but with free access to water. The extract was given orally before (prevention) or after (treatment) intragastric administration of absolute ethanol. Effects of dose (28 or 56mg/kg of body weight) and time of contact of the extract with gastric mucosa (1 or 2h) were also assessed. Animals were sacrificed, being the stomachs removed and the lesions were assessed by macroscopic observation and histopathology.C. citratus extract, given orally before or after ethanol, significantly (P0.01) reduced gastric mucosal injury compared with control group (vehicle+ethanol). The effect does not appear to be dose-dependent. Results also suggested that the extract is more effective when the time of contact with gastric mucosa increases.The results of this assay confirm the gastroprotective activity of C. citratus extract on experimental gastric lesions induced by ethanol, contributing for the pharmacological validation of its traditional use.

Iron-containing preparations available on the market vary in dosage, salt, and chemical state of iron contained in the preparation, as well as in the iron delivery process (immediate or prolonged-release). The present study aimed at characterizing the serum pharmacokinetics of iron in non pregnant women with iron deficiency anaemia (IDA) following a single oral administration of a prolonged-release ferrous sulphate tablet. This multicenter, single dose, open-label study was conducted in 30 women aged between 18 and 45 years with IDA. A single 160 mg oral dose of ferrous sulphate was given as 2 tablets of 80 mg of Tardyferon ® under fasting conditions. Blood samples were collected before dosing and until 24 h post-dosing. Serum iron concentrations were determined using a routine colorimetric analytical method. Pharmacokinetic parameters were determined from the serum concentration profiles using a non compartmental approach. Serum profiles showed elevated levels of iron up to 12 h after drug intake. The median time to maximum serum concentrations (T max ) occurred 4 h post-dosing. Between 2 and 8 h post-dosing, mean serum iron concentrations fluctuated by only 20%. Additionally, C 8h and C 12h represented on average 78.6% and 47.5% of the C max , respectively. This study demonstrates that a single oral dose of 160 mg Tardyferon ® administered under fasting condition to 30 women with IDA leads to an optimal long-lasting release of iron in the gastrointestinal tract in the targeted population. This allows the attainment and maintenance of elevated serum iron levels for up to 12 h after administration.

To optimize minituber production through aeroponics some horticultural management factors should be studied. Potato plantlets, cv Zorba, were grown aeroponically at two different plant densities (60 and 100 plants/m2). Plants showed an extended vegetative cycle of about 5 months after planting. A higher number of stolons was obtained at low plant densities. Tuber formation hastened when supplied N was reduced. Experiments on harvesting intervals (7, 10, and 14 days) indicated that for a density of 60 plants/m2, both number of minitubers and yield increased as harvesting interval decreased. Best results were achieved harvesting every 7 days: a total tuber yield of 118.6 g per plant was obtained (four times higher than for 100 plants/m2). Such a yield was composed, on the average, of 13.4 tubers with a mean tuber weight of 8.1 g. Harvesting intervals did not have an effect on the number of minitubers and yield for a density of 100 plants/m2. The best productivity obtained in this study was 800 minitubers/m2 for weekly harvests and a low plant density (60 plants/m2). We also studied the field performance of aeroponically produced minitubers vs those produced by hydroponics. Minituber behavior under field conditions was independent from the technique used for its production.

Study objectives To compare in a clinical setting the within-session reproducibility of two pulmonary function electronic monitoring devices (PiKo-1; Ferraris Respiratory Europe; Hereford, UK; and Spirotel; MIR; Rome, Italy) with one mechanical device (Mini-Wright Peak-Flow Meter; Clement-Clarke International; Harlow, Essex, UK), and to evaluate the accuracy of these devices using as reference an office pneumotachograph. Design, setting, and participants After detailed instructions, adults without airways diseases and patients with stable asthma attending an outpatient clinic performed four sets of expiratory maneuvers, one set for each device, in a strictly random order. Each set comprised three maneuvers with 2 to 3 min of rest between them. Measurements Reproducibility of FEV1 and peak expiratory flow (PEF) was assessed by a coefficient of variation (CV) and intraclass correlation coefficient (ICC), and accuracy was assessed by ICC and limits of agreement. Results Of the 38 participants evaluated, 71% were women and 61% had asthma. Ages ranged from 18 to 58 years, and FEV1 ranged from 1.2 to 4.8 L. In all monitoring devices, CV was 0.94 for the reproducibility of both FEV1 and PEF measurements. The accuracy of the PiKo-1 device was better for FEV1 (ICC = 0.98) than for PEF (ICC = 0.90). The Spirotel device had similar results for FEV1 and PEF (ICC = 0.95). The Mini-Wright device had the lowest accuracy (ICC = 0.87), particularly for PEF values Conclusions These low-cost and easy-to-use electronic monitoring devices showed a very good reproducibility and were in agreement with the pneumotachograph. Therefore, the PiKo-1 and Spirotel devices seem adequate for both screening and monitoring. However, prospective studies are still needed to assess their long-term reproducibility and usability and, particularly, the effects on the improvement of respiratory care.

The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant). Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg/g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg/g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G(M1)-ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (approximately 100 mg for a dose of 500 microg antigen) and would permit encapsulation of freeze-dried material or pill formation.

The gelling agent Phytagel™ was compared with Difco Bacto-agar forin vitro tuberization of six potato cultivars (Jaerla, Blanka, Claustar, Kennebec, Desiree, and Baraka). On a culture medium with 6% sucrose but lacking growth regulators, tuberization was higher when Phytagel™ was used rather than Difco Bacto-agar, regardless of the photoperiodic regime used. Chemical analyses of the gelling agents revealed a higher mineral content and organic impurities in Bacto-agar than in Phytagel™, which is therefore recommended for microtuber production. The same gelling agent should be used in all treatments of microtuberization experiments in order to draw meaningful physiological conclusions.

The main purpose of this study was to characterize the initial neurotransmission cascade elicited by methamphetamine, analysing simultaneously with in vivo microdialysis monoamine, amino acid and neuropeptide release in substantia nigra and neostriatum of the rat. The main effect of a single systemic dose of methamphetamine (15 mg/kg, subcutaneously) was an increase in dopamine levels, both in substantia nigra ( approximately 10-fold) and neostriatum ( approximately 40-fold), accompanied by a significant, but lesser, increase in dynorphin B ( approximately two-fold, in both regions), and a decrease in monoamine metabolites. A similar effect was also observed after local administration of methamphetamine (100 microm) via the microdialysis probes, but restricted to the treated region. In other experiments, rats were repeatedly treated with methamphetamine or saline, with the last dose administered 12 h before microdialysis. Dopamine K+-stimulated release was decreased following repeated methamphetamine administration compared with that following saline, both in the substantia nigra (by approximately 65%) and neostriatum (by approximately 20%). In contrast, the effect of K+-depolarization on glutamate, aspartate and GABA levels was increased following repeated administration of methamphetamine. In conclusion, apart from an impairment of monoamine neurotransmission, repeated methamphetamine produces changes in amino acid homeostasis, probably leading to NMDA-receptor overstimulation.

Complementary DNA expression of mature human serum albumin was engineered into potato plants under the transcriptional control of patatin B33 promoter and potato proteinase inhibitor II terminator. Protein secretion was achieved by using the signal sequence from potato proteinase inhibitor II. Recombinant albumin accumulated up to 0.2% of total soluble tuber protein in single transformant lines, regardless of the potato cultivar used. Electrophoretic mobility and N-terminal amino acid sequence analysis of partially purified recombinant albumin confirmed proper processing of an immune responsive recombinant albumin, and revealed that the proteinase inhibitor II signal sequence was correctly removed. No further optimisation of these yields was obtained by HSA expression in patatin antisense plants (line Pas58). Subcellular localisation showed that recombinant protein was successfully targeted to the apoplast. Potato tubers may be used, by applying this technology, to produce other heterologous proteins of interest in the biopharmaceutical industry.

SummaryThe maturity of a new potato clone can be determined from physiological measurements in field trials that involve considerable time, space and work. To improve the procedure, an in vitro bioassay based on tuberization behaviour of potato cultivars of different maturity classes was established. Twenty-six sets of temperature, photoperiod and photosynthetic photon flux values were assayed on the eight potato cultivars Jaerla, Zorba, Spunta, Kennebec, Turia, Desiree, Baraka and Fenix. In vitro tuberization was influenced by cultivar, photoperiod and temperature. The environmental condition defined by 12 h photoperiod, 34 μmol m–2 s–1 of photosynthetic photon flux and 25°C temperature produced statistical differences in tuberization among maturity classes. Both the earliness of tuberization and the “degree of tuberization” (based on the type of microtubers developed) permitted assigning a cultivar to a maturity class. Early cultivars showed higher readiness to tuberize and higher values of “degree of t...

Cardiac resynchronization therapy (CRT) is a new method for the correction of inter‐ and/or intraventricular conduction delays of patients with heart failure. The long‐term impact of CRT on central hemodynamics is not fully characterized. We performed complete right heart catheterization studies in 31 patients receiving a CRT device pre and 6 months after implantation. Most of the patients improved in their NYHA stage, their LVEF, and in parallel showed reduced right atrial (RA) pulmonary artery (PA) and pulmonary capillary wedge (PCW) pressures and pulmonary vascular resistance both at rest and at 25 watts. In addition, we found a reduction in heart rate accompanied by an increased mean arterial pressure both at rest and at 25 watts. Accordingly, brain natriuretic peptide levels (BNP) were lowered. It was concluded that, besides other well‐known effects on ventricular coordination, central hemodynamics after 6 months were improved during CRT. Copyright © 2007 Wiley Periodicals, Inc.

The tubby strain of mice exhibits maturity-onset obes-ity and sensory deficits in vision and hearing. The mu-tated gene, tub, responsible for this phenotype wasidentified recently, but the function of the TUB proteinhas not been deduced from its amino acid sequence.This prompted us to undertake expression mappingstudies with the hope that they might help to elucidatethe biological role of the TUB protein. We report the tubgene expression pattern in embryonic, fetal and adultmice tissues as determined by northern blots and insitu hybridization, using antisense oligonucleotidicprobes. In mouse embryos, tub is expressed selective-ly in differentiating neurons of the ensemble of centraland peripheral nervous systems, starting at 9.5 daysafter conception. In adult mice, tub is transcribed inseveral major brain areas, including cerebral cortex,hippocampus, several nuclei of the hypothalamuscontrolling feeding behavior, in the spiral ganglion ofthe inner ear and in the photoreceptor cells of the ret-ina. These structures contain potential cellular targetsof the tubby mutation-induced pathogenesis. Theneuronal-specific tub gene distribution allows theestablishment of a genotype–phenotype correlation inthe tubby mice. This correlation is reminiscent of thatobserved in fat/fat mice, whose phenotype, alsocharacterized by obesity, is caused by a null mutationin the carboxypeptidase E (CPE) gene. Our observa-tions highlight similarities between CPE, prohormoneconvertases, several neuropeptides and tub gene ex-pression patterns during embryogenesis, and maynarrow down the avenues to explore in order to deter-mine ultimately the function of the TUB protein. INTRODUCTIONThe tubby strain of mice exhibits an unusual phenotype whichcombines maturity-onset obesity and sensory deficits (1,2).Compared with the massive, early-onset obesity seen in ob/ob anddb/db mice, obesity in these mice develops more slowly and isless severe (1). The sensory defects in the tubby mice appearrestricted to vision and hearing. Previous observations (2,3)indicate that the tubby retina is characterized by a progressive lossof photoreceptors resulting in a reduced amplitude of theelectroretinogram which is extinguished by 6 months of age.Hearing loss is due to the degeneration of the organ of Corti andloss of afferent neurons in the basal end of the cochlea. Thesedeficits resemble those found in various human syndromesincluding Usher, Alstrom and Bardet–Biedl syndromes.The mutated gene responsible for the tubby phenotype wasidentified recently. A G →T transversion abolishes a donor splicesite in the 3′ coding region of this gene (4,5). This alterationresults in a larger than normal transcript containing an unsplicedintron, and the mutant mRNA is predicted to encode a truncatedprotein. The mouse tub gene belongs to a novel gene family,including three recently identified human genes: TUB, thehomologue of the mouse tub gene, and two related genes TULP1and TULP2 . The normal tub gene encodes a hydrophilic proteinthat lacks any known secretory sequence, mitochondrial transitsignal sequence, transmembrane domain, DNA- or RNA-bindingdomain. Recent data suggest that tub genes exist in a large numberof species, ranging from human to yeast (6). However, thefunction of the protein has not been deduced from knowledge ofits amino acid sequence. Consequently, we decided to undertakeexpression mapping studies with the hope that they might help usto understand the biological role of the TUB protein.We report here the tub gene expression pattern in embryonic,fetal and adult mice tissues as determined by northern blots andin situ hybridization. In embryos, tub is expressed selectivelythroughout the central and peripheral nervous systems indifferentiating neurons, starting at 9.5 days post-conception(d.p.c.). In adult mice, tub is transcribed in several major brainareas, including nuclei controlling feeding behavior, in the spiralganglion of the inner ear and in the photoreceptor cells of theretina. These structures contain potential cellular targets of thetubby mutation-induced pathogenesis. The strongly neuronal-specific tub gene distribution allows the establishment of agenotype–phenotype correlation in the tubby mice. This correla-tion is reminiscent of the one observed in fat/fat mice, whosephenotype, also characterized by obesity, is caused by a nullmutation in the carboxypeptidase E (CPE) gene (7). Our

Under the clinical impression that patients with chronic obstructive pulmonary disease (COPD) may demonstrate signs compatible with hypogonadism, we investigated whether oral glucocorticoid therapy is associated with testosterone deficiency. Thirty six men with COPD of whom 16 were receiving oral glucocorticoid medication (mean+/-SEM dose 9.4+/-1.1 mg prednisolone) were investigated in a cross-sectional cohort study. Patients with or without oral glucocorticoid therapy were not different in terms of age, smoking history and additional therapy. Vital capacity, forced expiratory volume in one second, airway resistance, intrathoracic gas volume and blood gases at rest were not different between the groups. However, patients receiving glucocorticoids had a shorter 6 min walking distance (mean+/-SEM 205+/-27 versus 288+/-26 m; p=0.02) compared to patients without oral steroid therapy. Serum levels of testosterone (mean+/-SEM 13.7+/-0.9) were below normal (

A medium containing low amounts of nitrogen (19–23 meq.l−1) produced optimum results in micropropagation as revealed by the number of nodes, internode length, chlorophyll content, and leaf area of four potato cvs. belonging each to four different maturity groups. Decreasing amounts of nitrogen also increased chlorophyll content in all four cultivars tested. The NH 4 + concentration did not have an effect on micropropagation for low nitrogen supplies. In all cvs., except Baraka, there was a “carry over” effect of the nitrogen content in the micropropagation medium onto subsequent tuberization, the lower nitrogen (23 meq.l−1) advancing tuber initiation. Microtuberization of cv. Jaerla was earlier in darkness than under short days regardless of the propagation medium used.

Summary A significant increase in the number of tubers was obtained in the presence of either kinetin (11.6·10 -3 mM), paclobutrazol (8.5·10 -3 mM) or acetate (5.4 mM) for in vitro stolons in darkness, and both single-node sections and intact 3-cm long sprouts were produced after long photoperiod (16 h) subculture. Longitudinal growth decreased whenever tuberization was promoted, especially in the presence of paclobutrazol. Calcium chloride (6 mM) slightly induced tuberization in the dark. Putrescine (0.15 mM) favoured vegetative development in all explants.

Recently the existence of a neurotensin striatonigral pathway strongly up-regulated by methamphetamine has been demonstrated. The aim of the present study was to investigate, using immunohistochemistry and radioimmunoassay, the modulation of this pathway by dopamine antagonists. Rats were injected either with methamphetamine alone or together with the dopamine D1 receptor antagonist, SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-be nzapine hydrochloride), or with the dopamine D2 receptor antagonist, sulpiride. Both techniques showed that this neurotensin striatonigral pathway is regulated by dopamine D1 receptors, since SCH 23390 totally prevented the methamphetamine-induced increase in neurotensin-like immunoreactivity, both in the striatum and in the substantia nigra pars reticulata. Conversely, sulpiride was unable to counteract the effect of methamphetamine in these two areas, suggesting that dopamine D2 receptors are not involved in the regulation of this neurotensin pathway. On the other hand, neurotensin-like immunoreactivity was markedly increased in striatal cell bodies and in the globus pallidus after treatment with sulpiride, indicating that this pathway is mainly regulated by dopamine D2 receptors.

Using immunohistochemical techniques we have analysed the occurrence of cholecystokinin-like immunoreactivity (CCK-LI) in the cortex and striatum of the rat. In the cortex few CCK-immunoreactive cell bodies, mainly interneurons, could be visualized in normal brains, and a moderately dense network of CCK fibres was also observed. Injections of colchicine into the striatum led to an accumulation, in the surrounding cortex, of CCK-LI in the initial segment of the axon of numerous cells. In addition, with an antibody to pro-CCK several cell bodies, many of which with pyramidal shape, could be visualized. Furthermore, retrograde staining of cortical cells after unilateral injection of wheat germ agglutinin into the striatum revealed bilaterally in the cortex a number of labelled cells that also contained pro-CCK-LI. In the striatum CCK-LI was diffusely distributed in fine fibres as well as in patches of fibres located in the medial aspects. After decortication followed by callosotomy these patches disappeared on the side ipsilateral to the lesion, while the pattern of immunoreactivity of several other peptides in the striatum was unaffected. No change was observed on the contralateral side. Decortication or callosotomy alone did not affect the pattern of CCK-LI. At the ultrastructural level several CCK-immunoreactive terminals could be observed, mostly with clear, densely packed vesicles and straight asymmetric synaptic contacts with small spines, characteristic for terminals of cortical origin. The results are consistent with the presence of a major, partly crossed, CCK-containing cortico-striatal pathway.

The effect of acute subcutaneous administration of methamphetamine on the expression of neurotensin mRNA was investigated in the adult rat striatum. At different time points (2, 6 and 24 h) following drug administration rats were killed, and mRNA levels were quantified both on films and emulsion-dipped tissue sections from two striatal levels. Two hours after methamphetamine injection, a dramatic increase in neurotensin mRNA levels was detected in different areas of the striatum at both rostral and caudal levels. Numerous positive cells were observed in the dorsomedial, dorsolateral and ventrolateral parts of the striatum. This up-regulation reflected an increase both in the number of cells expressing neurotensin mRNA and in the mean mRNA levels. This increase was still present after 6 h and was similar to the 2 h treated group at the rostral level of the striatum, but lower at the caudal one. Twenty-four hours after methamphetamine injection, neurotensin mRNA levels were back to control values, or in some areas even below. A strong increase in neurotensin mRNA-expressing cells was also seen in the olfactory tubercle, and the time-course was similar to the one observed in the striatum. In a second set of experiments, the effect of methamphetamine was evaluated on adjacent striatal sections hybridized with probes directed against neurotensin and substance P mRNAs, respectively. Two hours after drug administration, a significant increase in the levels of both peptide mRNAs was observed (+190% for neurotensin, +80% for substance P). These results demonstrate that methamphetamine is able to induce a dramatic, rapid and transient increase in striatal neurotensin mRNA levels, which may partly account for the elevation in neurotensin peptide levels observed in the striatonigral pathway after methamphetamine. The different anatomical localization of neurotensin mRNA-expressing cells observed after haloperidol and methamphetamine treatments, as well as the fact that the D1 receptor antagonist SCH-23390 is able to counteract the effect of methamphetamine but not that of haloperidol on neurotensin mRNA expression, suggests that there are at least two different subpopulations of neurotensin cells in the striatum. One population is regulated via D1 receptors and projects to the substantia nigra pars reticulata. The second is sensitive to D2 receptor stimulation and may project to the globus pallidus and/or may represent interneurons.

As recently described, adherence to the Mediterranean diet is associated with improved asthma control. However, evidence of how specific nutrients such as fatty acids and antioxidants may affect this relationship remains largely unknown. We aimed to examine the association between dietary intake of fatty acids and antioxidants and asthma control. A cross-sectional study was developed in 174 asthmatics, mean age of 40 (sd 15) years. Dietary intake was obtained by a FFQ, and nutritional content was calculated using Food Processor Plus™ software (ESHA Research, Inc., Salem, OR, USA). Good asthma control was defined by the combination of forced expiratory volume during the first second, exhaled NO (eNO) and Asthma Control Questionnaire (ACQ) score (control: forced expiratory volume in the first second ≥ 80 %; eNO ≤ 35 ppb; ACQ n-6:n-3 PUFA ratio predicted high eNO, whereas high intakes of n-3 PUFA, α-linolenic acid (ALA) and SFA were associated with low eNO. Odds for controlled asthma improved along with an increased intake of n-3 PUFA (OR 0·14, 95 % CI 0·04, 0·45; P for trend = 0·001), SFA (OR 0·36, 95 % CI 0·13, 0·97; P for trend = 0·047) and ALA (OR 0·18, 95 % CI 0·06, 0·58; P for trend = 0·005). A high n-6:n-3 PUFA ratio increased the odds for uncontrolled asthma (OR 3·69, 95 % CI 1·37, 9·94; P for trend = 0·009), after adjusting for energy intake, sex, age, education and use of inhaled corticosteroids. Higher intakes of n-3 PUFA, ALA and SFA were associated with good asthma control, while the risk for uncontrolled asthma increased with a higher n-6:n-3 PUFA ratio. The present results introduce a protective effect of ALA in asthma control, independent of marine n-3 fatty acids, and provide a rationale to dietary intervention studies in asthma.

Axonal transport of the vesicular monoamine transporter was assayed in the rat brain by in vivo binding of the specific ligand [3H]reserpine. Because of the marked localization of reserpine binding sites in dopaminergic cell bodies and nerve terminals, the dopaminergic nigrostriatal pathway was chosen for the study of the axonal transport of the monoamine carrier present in the membrane of synaptic vesicles. When labelled reserpine was injected into the substantia nigra, a delayed accumulation of radioactivity in the ipsilateral striatum was observed approximately 4 h after the injection. Similarly, injection into the right striatum was followed by a substantial accumulation of radioactivity in the ipsilateral substantia nigra, which was prevented by peripheral injection of unlabelled reserpine or tetrabenazine. This process was rapid and dependent on microtubules. In senescent rats, the amount of retrogradely transported [3H]reserpine was significantly reduced. These results demonstrate that labelled reserpine may be used to monitor in vivo fast axonal transport in central neurons.

Screening for allergic diseases allows an early diagnosis to be made, thus reducing socioeconomic burden and enhancing quality of life. We determined the prevalence of elevated exhaled nitric oxide (eNO) levels and sensitization to common airborne allergens in schoolchildren from different socioeconomic backgrounds.The study population comprised 271 children (136 boys) aged between 8 and 12 years in whom we applied skin prick tests and determined eNO levels.Thirty-five percent of the children were identified as atopic. There was a significantly higher prevalence of atopy in boys (43% vs. 27%). Among the children with a high socioeconomic status, 37% were atopic, compared with 30% of those with a low socioeconomic status. We observed a significantly lower prevalence of pollen sensitization in children of lower socioeconomic status (10% vs. 20%). In the atopic group, 51% had elevated eNO levels, while in the nonatopic group this value was 4%. The mean (SD) eNO level was 33.4 (26.6) ppb in the atopic group and 11.9 (6.4) ppb in the nonatopic group. No differences were found for eNO values in terms of gender or socioeconomic status.Our results emphasize the high prevalence of atopy in our population and suggest that determination of eNO levels could help to provide an early diagnosis. We also observed the following: (a) a significant difference in mean eNO values between atopic and nonatopic children; (b) a significantly lower prevalence of pollen sensitization in children of lower socioeconomic status; and (c) a higher prevalence of atopy in boys.