Measurement of yield and quality of DNA in human buffy coat is extraction method dependent.

During the last few years, an important element in the improvement of the molecular biology techniques has been the necessity for availability of high quality and functionality DNA. Several DNA extraction procedures with different results in both performance and quality, have been proposed. In this study our objective was to determine the most reliable extraction method that balances DNA quantity, and to assess the sample quantification of the fluorometric DNA quantification methods. For this, blood extracted by venopunction from 20 healthy volunteers was used to obtain DNA from buffy coat, and 4 commercial DNA extraction kits were assessed as well as two fluorometric DNA quantification methods with protocols of different complexity. Results suggest that manual methods achieve higher quality and larger yields of DNA. DNA purity obtained with the 4 extraction kits evaluated through the 260/280 and 260/230 ratio showed that the Qiacube kit fulfilled the criteria established in this work, followed very close by the Flexigene kit. On the other hand, the fluorometric DNA methods used in the samples quantification showed a higher variability when using QuantiFlour method, obtaining better results probably due to the simplicity of this protocol.

Features of aerobic granular sludge formation treating fluctuating industrial saline wastewater at pilot scale.

A pilot-scale sequencing batch reactor, with a working volume of 3 m3, was installed in a fish cannery to develop aerobic granular sludge treating the produced effluents. Depending on the nitrogen (N) and organic matter (COD) concentration, the effluents were named in this study as medium-low-strength (Stage I) and high-strength (Stage II) wastewater. The composition of the wastewater was found to be a crucial factor to select granule-forming organisms. With medium-low-strength wastewater as feeding, the first granules were observed after 30 days, but the extremely high COD/N ratios of the wastewater provoked the overgrowth of filamentous bacteria after 4 months of operation (Stage I). When treating high-strength wastewater, stable aggregates with good settleability appeared, but well-shaped granules were not observed since the granulation process was not completed. The system was able to remove both COD (70-95%) and N (30-90%) treating both types of effluents. Biomass growth was the main N removal pathway. The reactor was found to be robust against factory production stops and, thus, a suitable alternative to treat wastewater from industries with discontinuous operation.

Effect of marker dosage frequency and spot fecal sampling frequency in the prediction accuracy of fecal output using chromic oxide and titanium dioxide in grazing BON steers.

This study aimed to evaluate the effect of two marker dosage frequencies and two spot fecal sampling frequencies on the variability of fecal output estimation and fecal recovery rate in grazing Blanco Orejinegro (BON) steers with chromium (Cr) or titanium (Ti) as external markers. Four steers (230.5 ± 14.4 kg BW) were used in a 4 × 4 Latin square design in a split-plot arrangement with two markers: Cr or Ti, two marker dosage frequencies: 10.0 g Cr2O3 or TiO2/steer once daily (DF1) or 5.0 g Cr2O3 or TiO2/steer twice daily (DF2), and two fecal sampling frequencies: spot sampling once daily (SF1) or spot sampling twice daily (SF2). Steers were equipped with fecal collection bags to evaluate the actual fecal output (FOR). Fecal marker concentration (FMC) was affected by marker dosage frequency × fecal sampling frequency interaction (P = 0.032): SF1DF1 allowed higher FMC (2.26 g/kg DM) than SF2DF1 (1.95 g/kg DM). Chromium marker allowed higher estimated fecal output (FOe) values when compared to FOR (P < 0.001). Both Ti mean and SD bias (+ 0.074 ± 0.160 kg) were lower than Cr mean and SD bias (+ 0.312 ± 0.272 kg). The Ti fecal output prediction showed lower both RSR (0.586 vs 1.401) and MSPE (0.029 vs 0.167) than Cr prediction. The titanium external marker allowed fecal output estimation with more accuracy in grazing beef cattle trials. A protocol including a once-daily marker dosage associated with a once-daily fecal sampling is plausible, allowing good fecal recovery rates and accurate fecal output estimation.

Quality assessment on the long-term cryopreservation and nucleic acids extraction processes implemented in the andalusian public biobank.

Human samples are commonly collected and long-term stored in biobanks for current and future analyses. Even though techniques for freezing human blood are well established, the storage time can compromise the cell viability as well as the yield and quality of nucleic acids (RNA and DNA) extracted from them. In this study, a protocol to obtain peripheral blood mononuclear cells (PBMCs) from 70 subjects, which were stored at - 196 °C from EDTA tubes for a long-term, was assessed. In parallel; a protocol to obtain DNA from the same subjects, which were stored at - 80 °C from citrate tubes, was also studied. Samples stored from 2008 to 2012 were studied and the results obtained showed that there were no statistically significant differences in the RNA or DNA extracted in terms of purity, integrity and functionality The freezing protocol used by the Málaga Biobank shows that viable PBMCs and DNA could be kept for a period of, at least, 10 years, with a high quality and performance. Furthermore, RNA extracted from these PBMCs presents also a good quality and performance. Therefore, the samples frozen according to the conditions of the protocols assessed in this study could be optimal for biomedical research.

'What gets measured gets managed': revisiting the indicators for maternal and newborn health programmes.

BACKGROUND: The health of women and children are critical for global development. The Sustainable Development Goals (SDG) agenda and the Global Strategy for Women's, Children's, and Adolescent's Health 2016-2030 aim to reduce maternal and newborn deaths, disability, and enhancement of well-being. However, information and data on measuring countries' progress are limited given the variety of methodological challenges of measuring care around the time of birth, when most maternal and neonatal deaths and morbidities occur. MAIN BODY: In 2015, the World Health Organization launched Mother and Newborn Information for Tracking Outcomes and Results (MoNITOR), a technical advisory group to WHO. MoNITOR comprises 14 independent global experts from a variety of disciplines selected in a competitive process for their technical expertise and regional representation. MoNITOR will provide technical guidance to WHO to ensure harmonized guidance, messages, and tools so that countries can collect useful data to track progress toward achieving the Sustainable Development Goals. SHORT CONCLUSION: Ultimately, MoNITOR will provide technical guidance to WHO to ensure harmonized guidance, messages, and tools so that countries can collect useful data to track progress toward achieving the Sustainable Development Goals.

Morphometric study of the porcine placental vascularization.

The early development in mammals is characterized by the contribution of nutrients from the maternal tissues through the placenta, which is in apposition with foetal membranes and the endometrium, allowing the physiological interchange between the embryos/foetuses and the mother. The aim of this work was to study the number of placental blood vessels and their vascular area through morphometric analyses and the haemotrophic diffusion distance in porcine placental tissues from early gestations, intermediates gestations, advanced gestations and term gestations. For those purposes, morphometric measurements, blood vessel quantification, high-resolution light microscopy and transmission electron microscopy were performed. The implementation of the high-resolution light microscopy allowed studying the placental vascular and tissue histoarchitecture with higher definition and resolution than using a conventional light microscopy. We highlight the close location of the subepithelial capillaries to the maternal/foetal interface as pregnancy progresses. We found statistically significant evidence to state that the area of blood vessels is dependent on the gestation period. In advanced gestations, the presence of numerous small blood vessels and its near location to foetal/maternal interface agree with the great remodelling reported in our previous studies. In conclusion, in gilts, given the type of non-invasive epithelial placentation, the new blood vessels generation and of haemotrophic diffusion distance reduction, determined in this report, assure the maternal/foetal haemotrophic exchange efficiency during gestation.

Perceived efficacy and satisfaction of patients with subcutaneous hypoallergenic high-dose house dust mite extract.

SUMMARY: The efficacy and safety of subcutaneous immunotherapy with modified, high-dose, major allergen house dust mite extract is widely supported by double-blind, placebo-controlled studies. However, little is known regarding patient-perceived efficacy and satisfaction. An observational, retrospective, multicentre study in patients treated with Acaroid® was conducted to assess the efficacy and degree of satisfaction of the patients after the first six months of treatment with it. All the clinical study procedures were performed according to the routine clinical practice. This study demonstrates that Acaroid® is effective and well tolerated. The patients' condition demonstrated a clear and marked improvement in the first 6 months after treatment initiation. Patients treated with Acaroid® were very satisfied, with a correlation to improvement in patient-perceived symptoms and the administration of treatment by a healthcare professional.

[Early detection of lung cancer using computed tomography among patients with chronic obstructive pulmonary disease]. / Detección precoz de cáncer pulmonar con tomografía computarizada de tórax en pacientes con enfermedad pulmonar obstructiva crónica tabáquica.

BACKGROUND: Chest computed tomography (CT) scan may improve lung cancer detection at early stages in high risk populations. AIM: To assess the diagnostic performance of chest CT in early lung cancer detection in patients with chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: One hundred sixty one patients aged 50 to 80 years, active or former smokers of 15 or more pack-years and with COPD were enrolled. They underwent annual respiratory functional assessment and chest computed tomography for three years and were followed for five years. RESULTS: Chest CT allowed the detection of lung cancer in nine patients (diagnostic yield: 5.6%). Three cases were detected in the initial CT and six cases in follow-up scans. Most patients were in early stages of the disease (6 stage Ia and 1 stage Ib). Two patients were diagnosed at advanced stages of the disease and died due to complications of cancer. Two thirds of patients had nonspecific pulmonary nodules on the initial chest CT scan (100 patients, 62%). Seventy four percent had less than three nodules and were of less than 5 mm of diameter in 57%. In 92% of cases, these were false positive findings. In the follow-up chest CT, lung nodules were detected in two thirds of patients and 94% of cases corresponded to false positive findings. CONCLUSIONS: Chest CT scans may detect lung cancer at earlier stages in COPD patients.

Safety of cluster specific immunotherapy with a modified high-dose house dust mite extract.

INTRODUCTION: Although the efficacy and safety of high dose hypoallergenic mite subcutaneous immunotherapy (SCIT) using a conventional administration schedule has already been demonstrated, there is no reported experience on the safety of these extracts with cluster schedules. We wanted to determine whether the use of a cluster schedule of a hypoallergenic allergen with a high concentration of house dust mite allergens commonly used in normal practice was safe and well-tolerated in patients with dust mite allergy. MATERIAL AND METHODS: Multicentre, observational, retrospective study of dust mite allergic patients treated with a cluster schedule of SCIT (Acaroid; Day 1: 300/300 therapeutic units, TU--Day 8: 1000/1000 TU--Day 15: 3000/3000 TU) in 23 Spanish sites. RESULTS: Cluster schedule was used on 434 patients (40.1% children), with a total of 3256 doses (38.2% in children). There were 88 clinically relevant adverse reactions, 79 out of them local and 9 systemic (but mild-moderate) that amounted to 2.7% of all the administered doses. All the patients fulfilled the cluster schedule. CONCLUSIONS: Cluster schedule with high dose hypoallergenic mite-SCIT was safe and well-tolerated in routine clinical practice. Therefore, its use could reduce the costs and time needed to achieve the desired maintenance dose and increase compliance.

[Parkinson disease mortality rates in Chile]. / Mortalidad por enfermedad de Parkinson en Chile.

BACKGROUND: Parkinson disease (PD) is the second more common neurodegenerative disorder and determines a loss in quality of life, caregiver burden and increased mortality in those affected. AIM: To determine the rates of mortality due to Parkinson disease in Chile. MATERIAL AND METHODS: Data were collected from death certificates between 1997 and 2008. Mortality rates were calculated and the mortality trend was established along the study period, and analyzed by age, sex and geographic región of Chile. RESULTS: There was a steady increase in mortality over the years, particularly since 2001 (r²= 0.85, p < 0.01). The increase in mortality rates was 0.25 per 100.000 habitants/year (r²: 0.89, p < 0.01). Mortality was always higher in men and in those older than 80 years. The highest rate (per 100,000 habitants) in Chile was observed in the región of Valparaíso (2.4) while the lowest was in Antofagasta (0.8) (t = 3.72, p < 0.05). CONCLUSIONS: Mortality associated with PD has increased progressively, consistent with the expected increase in prevalence of the disease in the population.

Altitudinal variation in egg retention and rates of embryonic development in oviparous Zootoca vivipara fits predictions from the cold-climate model on the evolution of viviparity.

The evolution of reptilian viviparity is favoured, according to the cold-climate hypothesis, at high latitudes or altitudes, where egg retention would entail thermal benefits for embryogenesis because of maternal thermoregulation. According to this hypothesis, and considering that viviparity would have evolved through a gradual increase in the extent of intrauterine egg retention, highland oviparous populations are expected to exhibit more advanced embryo development at oviposition than lowland populations. We tested for possible differences in the level of egg retention, embryo development time and thermal biology of oviparous Zootoca vivipara near the extreme altitudinal limits of the species distribution in the north of Spain (mean altitude for lowland populations, 235 m asl.; for highland populations, 1895 m asl.). Altitude influenced neither temperature of active lizards in the field nor temperature selected by lizards in a laboratory thermal gradient, and pregnant females selected lower temperatures in the thermal gradient than did males and nonpregnant females across altitudinal levels. Eggs from highland populations contained embryos more developed at the time of oviposition (Dufaure and Hubert's stages 33-35) than eggs of highland populations (stages 30-34) and partly because of this difference incubation time was shorter for highland embryos. When analysed for clutches from both altitudinal extremes at the same embryonic stage at oviposition (stage 33), again incubation time was shorter for highland populations, indicating genuine countergradient variation in developmental rate. Our results indicate that temperature is an environmental factor affecting the geographical distribution of different levels of egg retention in Z. vivipara, as predicted by the cold-climate hypothesis on the evolution of viviparity.

Expression of myosin heavy chain isoforms in the human supraspinatus muscle: variations related to age and sex.

The contractile function of skeletal muscles is primarily regulated by the expression of myosin heavy chain (MHC) isoforms. Adult human skeletal muscles express three MHC isoforms (MHC-I, MHC-IIa and MHC-IIx). The muscles mainly expressing MHC-I are slow but resistant to fatigue, while those with major expression of MHC-IIa and MHC-IIx are fast and powerful but less resistant to fatigue. In this study, mRNA levels of the MHC isoforms were assessed in 24 human supraspinatus muscles by reverse-transcription polymerase chain reaction. The average expression of the MHC-I isoform was 36.72%, that of the MHC-IIa isoform was 33.52%, and the average expression of the MHC-IIx isoform was 29.76%. The higher average expression of the two MHC-II isoforms taken together (63.28%) indicates that the human supraspinatus muscle is a powerful, fast muscle with relatively low resistance to fatigue, in accordance with its role in the elevation of the upper extremity. In women, and more markedly in older women, the trend towards upregulation of the fast MHC-II isoforms and downregulation of the slow MHC-I isoform, which is absent in males, may improve our understanding of possible causes of the subacromial impingement syndrome.

Synthesis and characterization of PbSe nanoparticles obtained by a colloidal route using Extran as a surfactant at low temperature.

Lead selenide nanoparticles (PbSe NPs) have been obtained through an easy and low cost route using colloidal synthesis in aqueous solution. The synthesis was carried out at room temperature using Extran (Na5P3O10, NaOH and H2O) as surfactant. Hydrochloric acid (HCl) was used to eliminate the generated by-products. The size of PbSe NPs was varied by changing the Pb:Se molar concentration. The PbSe NPs were characterized by powder x-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDAX), high-resolution transmission electron microscopy (HRTEM) and Raman spectroscopy. The XRD measurements showed that the PbSe NPs have the face-centered cubic phase structure. The crystal size was found to be between 14 and 20 nm as calculated from the XRD patterns and these values were corroborated with SEM and TEM. Additionally, HRTEM micrographs showed crystalline planes at (200), (220) and (111) of the PbSe NPs, in agreement with the XRD results.

Development of a non-aqueous capillary electrophoresis method with UV-visible and fluorescence detection for phenolics compounds in olive oil.

Response surface methodology has been applied to the optimization of a simple and rapid non-aqueous capillary electrophoresis method for the separation and determination of several phenolic compounds belonging to the different families present in olive oil. A Box-Behnken design was employed and a total of 27 experiments were performed using olive oil samples spiked with the phenols and injected directly in the capillary after dilution 1:1 with 1-propanol. Finally, the background electrolyte (BGE) was constituted of 25 mM boric acid and 18 mM KOH in a mixture of 74:26 (v/v) 1-propanol/methanol. The hydrophobicity of the BGE allows its miscibility with the olive oil and, as a consequence, the possibility of characterizing and determining these kinds of compounds in this sample without any pretreatment. A hydrodynamic injection (6 s, -30 mbar) was applied and the separation was carried out using 35 °C and +20 kV of separation temperature and voltage, respectively. A capillary with two detection windows for serial online UV and fluorescence detection was satisfactorily employed. The validation of the method was carried out by setting the calibration curves, and the figures of merit were finally obtained. A lineal relationship between the corrected peak area and concentration and limits of detection in the order of micrograms per milliliter were found.

Ovarian response to oestrous synchronization protocol based on use of reduced doses of cloprostenol in cyclic goats.

The objective of this study was to assess the efficacy of two reduced doses vs a high/luteolytic dose of cloprostenol on luteolytic activity and synchronization of oestrus in cyclic goats. Experiment 1, included 24 goats randomly allocated to three groups: control group (group H) received a single high dose of cloprostenol (87.5 µg; 1.0 ml; i.m.) and M and L groups, which received half (43.75 µg; 0.5 ml) and a third (26.25 µg; 0.3 ml) of the highest dose, respectively. Experiment 2, included 24 goats randomly assigned to the same experimental groups. Each group was treated using two injections of cloprostenol administered 10 days apart to synchronize oestrus. Transrectal ultrasonographic scanning (US) was performed to detect the presence, size and development of corpora lutea and ovarian follicles. Furthermore, detection of oestrus was performed every 12 h between 24 and 72 h after the second injection of cloprostenol, and the luteolytic effect was verified by US. In Experiment 1, all goats that had corpora lutea at timing of treatment regressed their corpora lutea. In Experiment 2, the occurrence of oestrus and the interval between treatment to onset of oestrus were: 100%, 49.5 ± 3.0 h; 100%, 51.0 ± 3.0 h; and 75%, 56.0 ± 3.5 h for H, M and L groups, respectively. The development of preovulatory follicles and occurrence of subsequent corpora lutea were similar among groups. In summary, the use of 26.25 µg of cloprostenol is effective for the synchronization of oestrus in cyclic goats.

Follicular dynamics, interval to ovulation and fertility after AI in short-term progesterone and PGF2α oestrous synchronization protocol in sheep.

The study was aimed to assess the influence that short-term progesterone treatments have on follicular dynamics, oestrus and ovulation in sheep. The treatment was tested thereafter in a field trial to assess its fertility after AI with fresh semen. In a first experiment, 12 ewes without CL were grouped to receive a new (n = 6) or used CIDR (n = 6) for 7 days and blood samples were obtained to follow plasma progesterone profiles. In a second experiment, 39 cycling ewes were synchronized by a 7-day P4+PGF2α protocol using a new (n = 20) or a 7-day used CIDR (n = 19). Half of both groups received 400 IU eCG and half remained untreated as controls. Ultrasound ovarian examination and oestrous detection were used to compare follicular dynamics, oestrus and ovulation in both groups. In a third experiment, 288 ewes in 3 farms were synchronized by the short-term P4+PGF2α+eCG protocol and ewes were AI with fresh semen 24 h after oestrous detection. Lambing performance was used to test the fertility of the treatment. In Experiment 1, ewes with new inserts presented higher P4 concentration than ewes with used inserts throughout the sampling period (p < 0.05) and exhibited a P4 peak at days 1-2 of the treatment that was not observed in ewes with used inserts. In Experiment 2, ewes treated with new and used inserts show similar ovarian and behavioral traits (p > 0.10). However, ewes treated with eCG show shorter interval to oestrus (p = 0.004) and tend to have larger mature CL (p = 0.06). In Experiment 3, oestrous presentation and lambing performance after AI with fresh semen was considered normal compared to published results. Results suggest that the oestrous synchronization protocol based on P4+PGF2α allows little control of follicular dynamics without compromising fertility after AI with fresh semen provided that eCG is added at the end of the treatment.

[Outbreaks of acute gastroenteritis in Antofagasta Region, Chile 2010]. / Brote de gastroenteritis aguda en la Región de Antofagasta,Chile: 2010.

Outbreaks of acute gastroenteritis are a public health problem. Norovirus is known as the most common cause (50%). In Chile, immediate notification allows surveillance of these events. We describe an acute gastroenteritis outbreak that occurred in Antofagasta region, between March and April 2010. An observational study was conducted to perform the outbreak investigation. Local residents who met case definition were included. Stool samples, epidemiological surveys and environmental samples were requested. The outbreak began approximately on March 8, 2010 and lasted until April 28 with 31,036 reported cases (rate 54 per 1000 inhabitants). The most affected age group was between 25 and 44 years, and diarrhea was the main symptom (97% of cases). We determined the presence of norovirus genogroup II in clinical and environmental samples. This outbreak was caused by consumption of raw vegetables from La Chimba, which were watered and contaminated with treated sewage containing low concentration of free residual chlorine. Subsequently, the outbreak spread from person to person in a poor sanitary environment.

[Foodborne disease outbreaks around the urban Chilean areas from 2005 to 2010]. / Brotes de enfermedades transmitidas por alimentos y agua en la Región Metropolitana, Chile (2005-2010).

BACKGROUND: Foodborne disease outbreaks are one of the main health problems all over the world, which have an extensive impact on human health. OBJECTIVE: [corrected] To analyze the foodborne disease outbreaks occurred in Chilean urban area from 2005 to 2010. METHODS: We made a descriptive epidemiologic study. First, criteria were defined and classified according to previous epidemiologic investigations, clinical and environment samples, then. Variables of space, time, place and person were also analyzed. RESULTS: Among 2,806 reported outbreaks, 2434 (86.7%) fulfilled the inclusion criteria. Incidence rate of the period (2005-2010) were 32 cases per 100 inhabitants. A total of 12,196 people were affected, with an average of 5 patients per outbreak. The households (36.2%), restaurants (16.3%), supermarkets (6.3%) free fair (4.4%) have been the most important outbreak areas. The foods involved were seafood (15.4%), fish (15.1%), and fast food (13.5%). The etiologic agents were Salmonella spp, Shigella spp, Vibrio parahaemolyticus. CONCLUSIONS: Outbreaks foodborne diseases are frequents in the Chilean urban area, which make vulnerable a lot of people. The largest numbers happened in the households and were due to bad handling and/or inappropriate storage of the foods.