Cytogenetic relationships, polyploid origin and taxonomic issues in Paspalum species: inter- and intraspecific hybrids between a sexual synthetic autotetraploid and five wild apomictic tetraploid species.

Paspalum is a noteworthy grass genus due to the forage quality of most species, with approximately 330 species, and the high proportion of those that reproduce via apomixis. Harnessing apomictic reproduction and widening knowledge about the cytogenetic relationships among species are essential tools for plant breeding. We conducted cytogenetic analyses of inter- and intraspecific hybridisations involving a sexual, colchicine-induced autotetraploid plant of P. plicatulum Michx. and five indigenous apomictic tetraploid (2n = 40) species: P. compressifolium Swallen, P. lenticulare Kunth, two accessions of P. nicorae Parodi, P. rojasii Hack. and two accessions of P. plicatulum. Fertility of the hybrids was investigated and their reproductive system was analysed considering the relative embryo:endosperm DNA content from flow cytometry. Morphological, nomenclatural and taxonomic issues were also analysed. Cytogenetic analysis suggested that all indigenous tetraploid accessions of five apomictic species are autotetraploid or segmental allotetraploid. If segmental allotetraploids, they probably originated through autoploidy followed by diploidisation processes. Autosyndetic male chromosome pairing observed in all hybrid families supported this assertion. Allosyndetic chromosome associations were also observed in all hybrid families. In the hybrids, the proportion of male parent chromosomes involved in allosyndesis per pollen mother cell varied from 5.5% to 35.0% and the maximum was between 25% and 60%. The apomictic condition of the indigenous male parents segregated in the hybrids. These results confirm a strong association between autoploidy and apomixis in Paspalum, and the existence of cytogenetic relationships between different species of the Plicatula group. Allosyndetic chromosome pairing and seed fertility of the hybrids suggest the feasibility of gene transfer among species.

Preventing diarrhoea in enteral nutrition: the impact of the delivery set hang time.

BACKGROUND: To meet the current recommendations for enteral tube feeding (ETF), we updated our previous practice in 2011 and began to use a 24-h delivery set hang time (DSHT). We evaluated the impact of this update on the risk of diarrhoea and in diarrhoea-free survival. METHODS: Observational, retrospective study with historical controls on ischaemic and haemorrhagic stroke patients undergoing ETF. Diarrhoea occurrence (≥ 3 liquid stools in 24 h) was compared between patients with a 24 h DSHT (2011-2014) and a 72/96 h DSHT (2010-2011). The analysis was conducted using Kaplan-Meier curves and a Cox regression model. RESULTS: A total of 175 patients were included [median age 81 years (IQR = 12), 46.9% males], 103 in the group with a 24 h DSHT and 72 in the group with a 72/96 h DSHT. The group with a 24 h DSHT had a lower diarrhoea frequency (13.6% vs. 34.7%, risk ratio: 0.39, 95% CI: 0.22-0.70, p = 0.001) and a lower diarrhoea incidence rate (0.87 vs. 2.32 cases of diarrhoea/100 patient*day, rate ratio: 0.37, 95% CI: 0.19-0.72, p = 0.004). The Kaplan-Meier curves showed a longer diarrhoea-free survival for this group (p = 0.003, log-rank test). A 24 h DSHT was associated with a lower risk of diarrhoea (HR = 0.27, 95% CI: 0.12-0.61, p = 0.002), adjusted by albumin, stroke severity, intravenous thrombolysis, the administration of clindamycin and cefotaxime, and the administration of an enteral formula for diabetic patients. CONCLUSIONS: The 24 h DSHT was independently associated with a lower risk of diarrhoea and longer diarrhoea-free survival in hospitalised patients with acute stroke under ETF, compared with a 72/96 h DSHT.

Enteral tube feeding of patients with acute stroke: when does the risk of diarrhoea increase?

BACKGROUND/AIM: We aimed to evaluate the relationship between the length of time acute stroke patients underwent enteral tube feeding (ETF) and episodes of diarrhoea, and to investigate the temporal cut-off point at which diarrhoea risk increases. METHODS: An observational, retrospective study was conducted on patients with acute stroke admitted to a Stroke Centre. Patients undergoing ETF (ETF group) and those not undergoing ETF (control group) were analysed and matched by age and stroke severity. Data regarding demographic and clinical variables were recorded. The analysis was conducted using a receiver operating characteristic (ROC) curve and multivariate analyses. RESULTS: A total of 130 inpatients was included (age 75.08 ± 11.53 years, 56.2% men). The ETF group had higher diarrhoea frequency (27.7% vs 6.2%, P = 0.001). The length of time on ETF was associated with diarrhoea development (odds ratio (OR), 1.12 increment per day; 95% confidence interval (CI) 1.05-1.18; P < 0.001), after adjusting for confounders. The ROC curve showed 7 days on ETF as a cut-off point for diarrhoea risk. Seven days or more on ETF was independently associated with diarrhoea (OR, 6.26; 95% CI 1.66-23.62; P = 0.007), whereas less than 7 days was not when compared with the control group (OR, 0.38; 95% CI 0.04-3.91; P = 0.413). CONCLUSIONS: The length of time on ETF is associated with diarrhoea development in patients with acute stroke, demonstrating a temporal cut-off point. Seven days or longer on ETF is related to the occurrence of diarrhoea, whereas less than 7 days on ETF does not show this effect.

On-chip sample preparation and analyte quantification using a microfluidic aqueous two-phase extraction coupled with an immunoassay.

Immunoassays are fast and sensitive techniques for analyte quantification, and their use in point-of-care devices for medical, environmental, and food safety applications has potential benefits of cost, portability, and multiplexing. However, immunoassays are often affected by matrix interference effects, requiring the use of complex laboratory extraction and concentration procedures in order to achieve the required sensitivity. In this paper we propose an integrated microfluidic device for the simultaneous matrix clean-up, concentration and detection. This device consists of two modules in series, the first performing an aqueous two-phase extraction (ATPE) for matrix extraction and analyte pre-concentration, and the second an immunoassay for quantification. The model analyte was the mycotoxin ochratoxin A (OTA) in a wine matrix. Using this strategy, a limit of detection (LoD) of 0.26 ng mL(-1) was obtained for red wine spiked with OTA, well below the regulatory limit for OTA in wines of 2 ng mL(-1) set by the European Union. Furthermore, the linear response on the logarithmic concentration scale was observed to span 3 orders of magnitude (0.1-100 ng mL(-1)). These results are comparable to those obtained for the quantification of OTA in plain buffer without an integrated ATPE (LoD = 0.15 ng mL(-1)). The proposed method was also found to provide similar results for markedly different matrices, such as red and white wines. This novel approach based on aqueous two-phase systems can help the development of point-of-care devices that can directly deal with real samples in complex matrices without the need for extra extraction processes and equipment.

Aqueous two-phase systems for enhancing immunoassay sensitivity: simultaneous concentration of mycotoxins and neutralization of matrix interference.

Immunoassays have a broad application range, from environmental and food toxicology to biomedical analysis, providing rapid and simple methods for analyte quantification. Immunoassays, however, are often challenging at nM and sub nM concentrations and are affected by detrimental matrix interference effects, as is the case of the detection of ochratoxin A (OTA) and Aflatoxin B1 (AFB1). These are widespread mycotoxins found in food and feed, with serious potential implications for human health. This work demonstrates the use of polymer-salt aqueous two phase systems (ATPSs) for the simultaneous concentration of mycotoxins and neutralization of matrix interference. In particular, polyethylene glycol (PEG)-phosphate salt ATPSs were used to enhance the detection sensitivity of OTA and AFB1 in wines and beer by an indirect competitive ELISA. Using this methodology it was possible to quantify both analytes spiked in red wine with limits-of-detection (LoD) down to 0.19 ng/mL and 0.035 ng/mL, respectively, with results comparable to those obtained using solutions of toxins in phosphate buffered saline (PBS) buffer (0.7 ng/mL and 0.009 ng/mL, respectively). Furthermore, a very low matrix-to matrix variability was observed, with LoD and half inhibitory concentration (IC50) values of 5.17 ± 1.08 and 33.2 ± 3.5 ng/mL (±SD) obtained in the detection of OTA spiked in red and white wines, beer or PBS buffer. These results indicate the potential of ATPS as a fast and simple concentration step and in providing matrix-independent analyte quantification for enhanced immunoassay sensitivity below regulatory levels.

Integrated optical detection of autonomous capillary microfluidic immunoassays:a hand-held point-of-care prototype.

The miniaturization of biosensors using microfluidics has potential in enabling the development of point-of-care devices, with the added advantages of reduced time and cost of analysis with limits-of-detection comparable to those obtained through traditional laboratory techniques. Interfacing microfluidic devices with the external world can be difficult especially in aspects involving fluid handling and the need for simple sample insertion that avoids special equipment or trained personnel. In this work we present a point-of-care prototype system by integrating capillary microfluidics with a microfabricated photodiode array and electronic instrumentation into a hand-held unit. The capillary microfluidic device is capable of autonomous and sequential fluid flow, including control of the average fluid velocity at any given point of the analysis. To demonstrate the functionality of the prototype, a model chemiluminescence ELISA was performed. The performance of the integrated optical detection in the point-of-care prototype is equal to that obtained with traditional bench-top instrumentation. The photodiode signals were acquired, displayed and processed by a simple graphical user interface using a computer connected to the microcontroller through USB. The prototype performed integrated chemiluminescence ELISA detection in about 15 min with a limit-of-detection of ≈2 nM with an antibody-antigen affinity constant of ≈2×10(7) M(-1).

Precisión diagnóstica de un dispositivo automático en la determinación del índice tobillo-brazo en pacientes con ictus isquémico / Diagnostic accuracy of an automatic blood pressure device for ankle brachial index determination in ischemic stroke patients

Introducción. El uso del índice tobillo-brazo (ITB) no está extendido en la práctica clínica por la necesidad de equipo y formaciónespecíficos. Poder realizarlo sin estos requisitos, en menos tiempo y con menor coste, facilitaría su uso habitual. Objetivo. Evaluar el rendimiento de un esfigmomanómetro automático en la identificación de pacientes con un ITB < 0,90, cuando la técnica es realizada por diplomados en Enfermería. Secundariamente, evaluar la concordancia de esta técnica con el método de referencia.Pacientes y métodos. Estudio de precisión diagnóstica en 30 pacientes con ictus isquémico. En cada paciente se realizarontres determinaciones del ITB con un esfigmomanómetro automático y una con el método de referencia.Resultados. El rendimiento del método automático en la detección de ITB < 0,90 obtuvo valores aceptables en las tres determinaciones (sensibilidad: 78%, 87% y 100%; especificidad: 95%, 100% y 100%; valor predictivo positivo: 87%, 100% y100%; valor predictivo negativo: 91%, 96% y 100%; exactitud: 90%, 97% y 100%). Los cocientes de correlación intraclase intraobservador, interobservador e intermétodo del dispositivo automático presentaron niveles aceptables, con resultadosde 0,64, 0,83 y 0,75, respectivamente. En el test de Bland y Altman sólo la comparativa intermétodo mostró menosdel 5% de los valores fuera de rango. Conclusión. El rendimiento del esfigmomanómetro Omron HEM-907 ® en la identificación de pacientes con un ITB < 0,90presenta valores aceptables cuando la técnica es realizada por diplomados en Enfermería. Esto sugiere su posible utilidadcomo herramienta de cribado en pacientes con ataque isquémico transitorio/ictus isquémico (AU)

Introduction. Ankle brachial index (ABI) is not widespread in clinical practice because the need for specific equipment and training. Doing it without these requirements, quicker and with a cheaper method would facilitate their routine use.Aims. To evaluate sensitivity and specificity of an automatic blood pressure device detecting ABI < 0.90, when technique is performed by nurses. To evaluate agreement between two techniques for the ABI determination Patients and methods. Diagnostic accuracy study in 30 ischemic stroke patients. For each patient, three measurementswere performed with the automatic device, and one with the reference method. Results. The automatic blood pressure device performance detecting ABI < 0.90 was acceptable for each of the threemeasurements (sensitivity: 78%, 87% and 100%; specificity: 95%, 100% and 100%; positive predictive value: 87%, 100% and 100%; negative predictive value: 91%, 96% y 100%; accuracy: 90%, 97% y 100%). The intraclass correlation coefficients for intra-observer, inter-observer and inter-method variability showed acceptable levels, with results of 0.64, 0.83 and 0.75, respectively. In the Bland Altman test only inter-method comparison had less than 5% of its values out of range. Conclusions. Sensitivity and specificity of an automatic blood pressure device detecting ABI < 0.90 shows acceptable levels when technique is performed by nurses. This suggests its potential as screening tool in transient ischemic attack/ischemicstroke patients (AU)

Effect of charcoal types and grilling conditions on formation of heterocyclic aromatic amines (HAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled muscle foods.

Grilling muscle foods involves high temperatures that lead to production of cooking toxicants, such as heterocyclic aromatic amines (HAs) and polycyclic aromatic hydrocarbons (PAHs). To obtain realistic exposure levels of these two groups of mutagens analyses of the same samples using similar separation/detection techniques were performed. HAs and PAHs were quantified in well-done meat and fish samples grilled with wood and coconut shell charcoal at 200°C. Quantitative HAs and PAHs profiles were different for beef and salmon using the same type of charcoal. Higher levels of HAs and PAHs were found in salmon samples. No significant differences were observed for HAs and PAHs in beef samples grilled with both charcoal types, whereas salmon grilled with coconut shell charcoal presented significantly lower amounts of HAs and PAHs than salmon grilled with usual wood charcoal. Continuous barbecuing with the same charcoal shown that combustion of fat that dropped along the grilling period contributed to higher formation of HAs and PAHs. Special attention must be given to the intake of barbecued foods since high amounts of HAs and PAHs can be taken in a single meal.

A comparison of the extraction procedures and quantification methods for the chromatographic determination of polycyclic aromatic hydrocarbons in charcoal grilled meat and fish.

A method for analysis of 15 PAHs in charcoal-grilled meat/fish was established by high performance liquid chromatography and fluorescence detection. Gradient elution was performed with methanol/water/ethyl acetate. Maxima excitation and emission wavelengths were selected for each PAH. Retention times were very stable with coefficients of variation below 0.24% within analytical day and below 0.60% across analytical days. Two different methods of cleanup and pre-concentration steps were compared. Solvent extraction assisted by sonication carried out with n-hexane on 2g of lyophilized meat or 1g of lyophilized fish allowed to obtain high sensitivity, reproducibility and better extraction efficiency. Limits of quantification (LOQs, s/n=10) were lower than 0.01ng/g of meat wet weight and lower than 0.02ng/g of fish wet weight for all PAHs (except for Na, Fl and IP that were lower than 0.1ng/g). Two different quantification methods were compared. Standard addition method compensated PAHs losses due to incomplete extraction and it is recommended for analyses of grilled meat and fish samples that usually contain very low amounts of the eight high molecular weight PAHs (BaA, Ch, BbF, BkF, BaP, IP, BgP, DhA).

Heterogeneous immunoassays in microfluidic format using fluorescence detection with integrated amorphous silicon photodiodes.

Miniaturization of immunoassays through microfluidic technology has the potential to decrease the time and the quantity of reactants required for analysis, together with the potential of achieving multiplexing and portability. A lab-on-chip system incorporating a thin-film amorphous silicon (a-Si:H) photodiode microfabricated on a glass substrate with a thin-film amorphous silicon-carbon alloy directly deposited above the photodiode and acting as a fluorescence filter is integrated with a polydimethylsiloxane-based microfluidic network for the direct detection of antibody-antigen molecular recognition reactions using fluorescence. The model immunoassay used consists of primary antibody adsorption to the microchannel walls followed by its recognition by a secondary antibody labeled with a fluorescent quantum-dot tag. The conditions for the flow-through analysis in the microfluidic format were defined and the total assay time was 30 min. Specific molecular recognition was quantitatively detected. The measurements made with the a-Si:H photodiode are consistent with that obtained with a fluorescence microscope and both show a linear dependence on the antibody concentration in the nanomolar-micromolar range.

Extrahepatic immunological manifestations of hepatitis C virus in dialysis patients.

BACKGROUND: Hepatitis C virus (HCV) infection may be associated with various extrahepatic immunological disorders. Uremic patients on chronic regular dialytic treatment (RDT) frequently develop immunological abnormalities. The aim of this study was to evaluate the probability that HCV infection creates an increased risk for extrahepatic immunological abnormalities in chronic RDT patients. SUBJECTS AND METHODS: In a series of one hundred sixteen chronic RDT patients, HCV status was determined by anti-HCV antibodies, polymerase chain reaction (PCR) RNA and viral genotyping. After excluding four anti-HCV negative/PCRRNA positive patients, a comparison was made between 51 anti-HCV negative/PCR-RNA negative and 61 anti-HCV positive patients, this latter group including seventeen PCR-RNA negative, fifteen genotype 1, thirteen genotype 2, three genotype 3, four genotype 4, four undeterminable genotype and five mixed genotypes. The following investigations were performed: cryoglobulinemia (presence, titer and, when possible, identification), monoclonal gammopathy, antineutrophil cytoplasm antibodies, antidouble stranded DNA antibodies, circulating immunocomplexes and immunoglobulin levels. RESULTS: Cryoglobulinemia was found in 77% of anti-HCV positive versus 29% of anti-HCV negative patients, and cryocrit > 1% in 50% versus 9.8% respectively, p=<0.01. Also cryoglobulin concentration was higher (logarithmic transformation: 4.38 +/- 0.94 vs 3.11 +/- 1.06, p =< 0.001) in anti-HCV positive versus negative patients. Multivariate logistic regression analysis showed a significantly increased odds ratio (12.0, confidence interval 3.0 to 48.3) for having high levels of cryoglobulins (cryocrit >1%) after adjusting for age and dialytic age. The prevalence of this abnormality did not differ significantly among patients infected with different genotypes, but a tendency towards a lower frequency was observed in the anti-HCV positive/PCR negative subgroup. Cryoglobulins were identified as type I (2 anti-HCV positive case), type II (2 anti-HCV positive and 1 anti-HCV negative case) and type 3 (1 anti-HCV negative case). The frequency of monoclonal gammopathy was not significantly different between anti-HCV positive and anti-HCV negative patients (6.5% versus 2%) as well as that of the other parameters evaluated except for IgG concentration which was higher in the anti-HCV positive group (1,685 +/-605 versus 1349 +/- 352 mg/dl, p 0.006). Five events, potentially linked to HCV infection, occurred in our anti-HCV positive patients: 2 cases of porphyria cutanea, 1 case of unexplained peripheral neuropathy, 1 cutaneous leukocytoclastic vasculitis, 1 death for non-Hodgkin's lymphoma. In one anti-HCV positive patient treated with interferon-alpha, the presence of cryoglobulins, monoclonal gammopathy and high IgG levels strictly paralleled that of viremia, disappearing during the recovery phase under treatment and reappearing shortly after stopping treatment. CONCLUSIONS: HCV infection provides a significantly increased risk for developing extrahepatic immunological abnormalities also in chronic RDT patients. It is possible that the clinical relevance of this event might be scant because of the low level of these abnormalities, but an awareness of its possibility should to be taken into account.