Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Traumatic brain injury in the new millennium: new population and new management.

INTRODUCTION: Traumatic brain injury (TBI) is one of the leading causes of death and disability globally. We present a study describing epidemiological changes in severe TBI and the impact these changes have had on management and analysing alternatives that may improve outcomes in this new population. MATERIALS AND METHODS: We performed a retrospective, descriptive, cross-sectional analysis of patients presenting severe TBI at our hospital in the period of 1992-1996 and 2009-2013. We analysed demographic data, including age, sex, mortality, aetiology, anticoagulation, treatment, and functional outcome. RESULTS: We reviewed data from 220 patients. In the second cohort, there were 40% fewer patients, mean age was 12 years older, patients were more frequently receiving anticoagulation therapy, and the percentage of interventions was halved. Aetiology varied, with traffic accidents being the main cause in the first group, and accidental falls and being hit by cars in the second group. There were no intergroup differences for mortality or functional outcomes. CONCLUSION: The age of patients admitted due to severe TBI has increased. As a result of this, the main cause of severe TBI in our population is accidental falls in elderly, anticoagulated patients. Despite the low-energy nature of trauma, patients in the second cohort presented a poorer baseline status, and were less frequently eligible for surgery, with no improvement in mortality or functional outcomes.

Traumatic brain injury in the new millennium: A new population and new management. / El traumatismo craneoencefálico severo en el nuevo milenio. Nueva población y nuevo manejo.

INTRODUCTION: Traumatic brain injury (TBI) is one of the leading causes of death and disability globally. We present a study describing epidemiological changes in severe TBI and the impact these changes have had on management and analysing alternatives that may improve outcomes in this new population. MATERIALS AND METHODS: We performed a retrospective, descriptive, cross-sectional analysis of patients presenting severe TBI at our hospital in the period of 1992-1996 and 2009-2013. We analysed demographic data, including age, sex, mortality, aetiology, anticoagulation, treatment, and functional outcome. RESULTS: We reviewed data from 220 patients. In the second cohort, there were 40% fewer patients, mean age was 12years older, patients were more frequently receiving anticoagulation therapy, and the percentage of interventions was halved. Aetiology varied, with traffic accidents being the main cause in the first group, and accidental falls and being hit by cars in the second group. There were no intergroup differences for mortality or functional outcomes. CONCLUSION: The age of patients admitted due to severe TBI has increased. As a result of this, the main cause of severe TBI in our population is accidental falls in elderly, anticoagulated patients. Despite the low-energy nature of trauma, patients in the second cohort presented a poorer baseline status, and were less frequently eligible for surgery, with no improvement in mortality or functional outcomes.

Airflow obstruction case finding in community-pharmacies: a novel strategy to reduce COPD underdiagnosis.

BACKGROUND: Community pharmacies (CP) have access to subjects at high-risk of suffering Chronic Obstructive Pulmonary Disease (COPD). We investigated if a COPD case finding program in CP could be a new strategy to reduce COPD underdiagnosis. METHODS: Prospective, cross-sectional, descriptive, uncontrolled, remotely supported study in 100 CP in Barcelona, Spain. Pharmacists were trained in a four-day workshop on spirometry and COPD, and each was provided with a spirometer for 12 weeks. The program included questionnaires and forced spirometry measurements, whose quality was controlled and monitored by web-assistance. FINDINGS: Overall 2295 (73.5%), of 3121 CP customers invited to participate in the program accepted, and 1.456 (63.4%) were identified as "high risk" for COPD using the GOLD questionnaire. Only 33 could not conduct spirometry, and a pre-bronchodilator airflow limitation (FEV1/FVC ratio <0.7) was confirmed in 282 (19.8%); 244 of these were referred to their primary care (PC) physician for further diagnostic and therapeutic work-up, but only 39 of them (16%) fed-back this information to the pharmacist. Clinically acceptable quality spirometries (grade A or B) were obtained in 69.4% of the cases. CONCLUSION: This study shows that adequately trained and supported community pharmacists can effectively identify individuals at high risk of having COPD and can thus contribute to ameliorate underdiagnosis in this disease. Links between PC and CP should be improved to achieve a useful program.

COPD case finding by spirometry in high-risk customers of urban community pharmacies: a pilot study.

BACKGROUND: COPD case finding is currently recommended at primary and tertiary care levels only. AIM: To evaluate the feasibility of a community pharmacy program for COPD case finding in high-risk customers by means of spirometry. METHODS: Pilot cross-sectional descriptive study in 13 urban community pharmacies in Barcelona, Spain, from April to May 2007. Customers >40 years old with respiratory symptoms and/or a history of smoking were invited to participate in the study during pharmacists' routine work shifts. High-risk customers were identified by means of a 5-item COPD screening questionnaire based on criteria of the Global Initiative for Chronic Obstructive Lung Disease, and were invited to perform spirometry accordingly. Those with an FEV(1)/FVC ratio less than 0.70 were referred to the hospital for a repeat spirometry. RESULTS: Of the 161 pharmacy customers studied, 100 (62%) scored 3 or more items in the COPD screening questionnaire, and after spirometry, 21 (24%) had an FEV(1)/FVC ratio<0.7. When these subjects with airflow limitation were offered referral to a hospital respiratory function laboratory for further assessments, 11 (52%) attended the appointment. Over 70% of spirometries were rated as being of acceptable quality. No significant differences were observed in lung function parameters between the pharmacy and hospital measurements. CONCLUSIONS: COPD case finding by spirometry in high-risk customers of urban community pharmacies is feasible. Similarly to primary care practitioners, pharmacists have access to high-risk, middle-aged subjects who have never been tested for COPD. Pharmacists can help with early detection of COPD if they are correctly trained.

Tibial muscular dystrophy with late adult onset in a Spanish family.

PURPOSE: We report autosomal dominant distal muscular dystrophy in 5 members of a Spanish family. INTRODUCTION: This unusual muscular disorder has late adult onset and predominantly it affects the anterior compartment of the legs. This myopathy presented clinical and electromyographical characteristics, but unspecific histological findings. Early there have appeared genetical studies, the most frequently used is chromosome linkage, but it is not an absolute criterion for diagnosis, and it is not available in most hospitals. PATIENTS DESCRIPTIONS: In our cases walking difficulties appeared between the fourth and fifth decades, characterized by progressive and varied weakness with amyotrophy in the tibial anterior compartment. The electromyography confirmed the presence of a severe non-inflammatory myopathy, chronic and symmetric in the pretibial muscles and of less intensity in the calf muscles. The levels of creatine phosphokinase were normal and muscle biopsy identified a chronic, unspecific lesion with important fibrosis. CONCLUSIONS: The findings, although with some phenotypical differences, were those commonly found in Markesbery-Griggs disease, tibial muscular dystrophy or late onset type 2 distal myopathy. We report a family affected by this muscular disorder, we describe the differential diagnosis and we discuss the review of the available literature.

Lung cancer pulse oximetry and capnography in lung function laboratories.

OBJECTIVE: To compare values reflecting oxyhemoglobin saturation obtained by pulse oximetry (SpO2) and values for end-tidal carbon dioxide pressure (PETCO2) obtained by capnography with direct measures of gas saturation values and pressures (PaO2 and PaCO2) in arterial blood gas samples. PATIENTS AND METHODS: We studied 57 consecutive patients ranging in age from 34 to 83 years--16 of whom were smokers--who presented for lung function testing. RESULTS: The mean (SD) SpO2 was 95% (2.4%), and oxygen saturation measured directly in arterial blood samples was 95.1% (2.3%) (P=NS). The mean PETCO2 was 37.9 (5.3) mm Hg and PaCO2 by arterial blood gas analysis was 40.6 (5.4) mm Hg (P<.0001). The correlation between the 2 measurements of oxygen saturation (SpO2 and direct assessment) was 0.806 (P<.0001), and the correlation between PETCO2 and PaCO2 was 0.845 (P<.0001). The mean difference between the 2 expressions of oxygen saturation was 0.08% (1.46%) and between PETCO2 and PaCO2 was 2.7 (2.9) mm Hg. CONCLUSION: Both measurement devices (pulse oximeter and capnograph) are appropriate for use in a lung function laboratory. The difference between PETCO2 and the PaCO2 should be kept in mind.

Lack of effects of moderate-high altitude upon lung function in healthy middle-aged volunteers.

This study investigates the effects of moderate-high altitude on lung function and exercise performance in 46 volunteers (19 females, 27 males), with a mean age of 42.4 +/- 1.4 years (+/- SEM) and varying smoking and exercise habits, who were not previously acclimatized. Measures obtained in the base camp (1140 m) and at altitude (2630 m), in random order, included forced spirometry, maximal voluntary ventilation, maximal inspiratory and expiratory pressures, arterial oxygen saturation and capillary lactate concentration after a standardized exercise test. The smoking history, Fagerström test and degree of habitual physical activity were also recorded for each participant. The percentage of smokers was similar in males (19%) and females (21%) (P = n.s.). Mean habitual physical activity index was 8.2 +/- 0.2 (range, 5.88-11.63). At the base camp, all lung function variables were within the normal range. Lactate concentration after exercise averaged 3.7 +/- 0.3 mm l-1. No significant change was observed at altitude, except for a higher heart rate and a lower arterial oxygen saturation (SaO2) (both at rest and after inspiratory manoeuvres). The smoking history and the degree of physical activity did not influence lung function or exercise performance at altitude. The results of this study show that in middle-aged, healthy, not particularly well-trained individuals, lung function is not significantly altered by moderate-high altitude, despite the absence of any acclimatization period and independent of their smoking history and previous exercise habits.

[Induced sputum in asthma: study of validity and repeatability]. / El esputo inducido en el asma: estudio de validez y repetibilidad.

To assess the validity and reproducibility of determining total and differential cell (DC) counts and of eosinophilic cationic protein (EPC) levels in induced sputum. Clinical analysis of validity and reliability of a measurement tool. Twenty-one asthmatics [age: 31(14) years; FEV1: 78(21)% of reference value; PC20FEV1: 0.02(1.1)mg/ml] and 10 healthy subjects [age: 30(3) years, FEV1: 99(12)% of reference value, PC20FEV1: > 8(3.7)mg/ml]. A sputum sample was collected from each individual using hypertonic saline. Sputum was separated from saliva and then divided into two portions as uncontaminated as possible by squamous cells. Both portions were treated independently and simultaneously within two hours with a solution of bithiothreitol to disperse the cells, and then centrifuged. The supernatant was poured off and frozen al -70 degrees for later determination of EPC. The sediment was stained with Papanicolaou stain, toluidine blue and eosinhematoxylin. DC count was expressed as a percentage of total inflammatory cells. ECP was determined with a CAP-System and a commercial kit (Pharmacia Diagnostics SA, Uppsala, Sweden). Two subjects in each group were unable to produce valid sputum samples (20% of healthy subjects and 10% of asthmatics). The validity of the method was demonstrated by significant differences between asthmatics and healthy subjects in both eosinophil and EPC levels. The reproducibility of method was verified by comparing the results for the sputum fractions; no significant differences were found in cell counts or EPC levels. The within-group correlation coefficients for the sputum fractions ranges between 48 and 77% for all cell types, except squamous cells, which gave a coefficient of 18%. For EPC the correlation coefficient was 98%. DC and EPC determinations in sputum induced by hypertonic saline are valid and reproducible.

Evidence for a direct action of GH on haemopoietic cells of a marine fish, the gilthead sea bream (Sparus aurata).

Receptors for GH were characterized in the head kidney of gilthead sea bream (Sparus aurata), using radioiodinated and biotinylated ligands. The specific binding of radiolabelled recombinant gilthead sea bream GH (rsbGH) to head kidney membrane preparations was dependent on membrane concentration. Salmon prolactin, salmon gonadotrophin and carp gonadotrophin did not compete for 125I-labelled rsbGH-binding sites. Unlabelled rsbGH competitively displaced 125I-labelled rsbGH bound to head kidney membranes. Scatchard plots were always linear, denoting the presence of a single class of binding sites. The binding affinity (Ka = 2.7 x 10(9) M-1) was equivalent to that found in liver membrane preparations, but the binding capacity (2.5 +/- 0.30 fmol/mg protein) was 50- to 75-fold lower. To identify the cells which express the GH receptor, head kidney smears were incubated with biotinylated rsbGH, followed by incubation with an avidin-biotin complex conjugated to alkaline phosphatase. The reaction with the new-fuchsin substrate gave a red precipitate, showing a specific and intense labelling in erythroblasts, polychromatophilic erythroblasts and myeloblasts. Noticeable binding was observed in myelocytes and immature granulocytes, tending to disappear at the latter stages of granulocyte maturation. Light but appreciable binding was also observed in monocytes, lymphocytes and acidophilic erythroblasts, whereas it was completely absent in proerythrocytes and erythrocytes. The proliferative action of rsbGH and recombinant human IGF-I on in vitro cultures of head kidney cells was demonstrated by a 5-bromo-2'-deoxy-uridine immunoassay. To our knowledge, this is the first report that provides suitable evidence for a role of GH as a haemopoietic growth and differentiation factor in lower vertebrate species.

Novel methyltransferase activity modifying the carboxy terminal bis(geranylgeranyl)-Cys-Ala-Cys structure of small GTP-binding proteins.

Proteins containing CX3, CXC, and CC (where C is cysteine and X is undefined) undergo posttranslational isoprenylation at their cysteine residues. In the case of proteins which terminate in CX3, proteolytic removal of X3 is followed by the carboxymethylation of the isoprenylated cysteine residue. CXC proteins also undergo C-terminal methylation. The present study addresses the question of whether this methylation is catalyzed by a different isoprenylated protein methyltransferase than that previously described for CX3 proteins. The S-adenosylmethionine (AdoMet) dependent methylation of a small peptide-N-acetyl-S-geranylgeranyl-L-cysteinyl-L-alanyl-S-geranylgeranyl- L- cysteine (Ac(GG)CysAla(GG)Cys)--was investigated using membranes from a variety of bovine tissues as sources of enzyme. Ac(GG)CysAla(GG)Cys was a substrate for methylation, while Ac(GG)Cys(GG)Cys was not. Reciprocal inhibition studies on the methylation reactions of the CXC peptide and of N-acetyl-S-farnesyl-L-cysteine (AFC), a previously described methyltransferase substrate, suggested that these reactions are catalyzed by distinct enzymatic activities. Farnesylthioacetic acid (FTA), a potent competitive inhibitor of the methylation of AFC, did not inhibit the methylation of the CXC peptide. Moreover the KI values for S-adenosylhomocysteine and S-adenosylethionine inhibition differed for the two enzymatic activities. These data indicate that more than one AdoMet-dependent methyltransferase is involved in the carboxymethylation of isoprenylated proteins.

[Arterial puncture in Spain. A follow-up of the SEPAR 1987 guideline. Sociedad Española de Neumología y Cirugía Torácica]. / La punción arterial en nuestro ámbito. Seguimiento de la normativa SEPAR 1987.

This study sought to evaluate the procedures used for measuring arterial gases in our hospitals and determine the level of compliance with the 1987 SEPAR guidelines. Questionnaires covering the obtention, transport, storage and analysis of samples, as well as familiarity with the guidelines were distributed to 150 pneumology, internal medicine, intensive care and emergency services; 78 centers responded (71% from pneumology departments). General information obtained was as follows: arterial puncture is carried out by nurses without anesthesia and using syringes for gases; the radial artery is used; with post-puncture pressure provided by the patient; the sample is analyzed within 15 minutes and cold storage is used. The department's own automatic analyzer is used. Buffered gases and solutions are used for calibration and quality control measures are rare. Some aspects of the guidelines are unfamiliar. We believe that an effort should be made to extend application of the guidelines in order to improve arterial blood gas analysis.

Serum lipid levels before and after vasectomy in men.

The blood lipid profile was determined in sixty-two men, 24 to 62 years old, before and two, six and twelve months after surgical occlusion of the vas deferens. No statistically significant differences were found in mean body weight, blood pressure, serum levels of non-esterified fatty acids, total lipids, triglycerides, total cholesterol and alpha, beta and prebeta fractions of the lipoproteins, which were measured before and after surgery. When the serum levels of the alpha and beta fractions were considered in the same subject, it was observed that 12 months after vasectomy a similar percentage of cases showed a predominance of either one of them. Hence, no modifications on the lipid profile of these subjects were found that could indicate an increased risk of arteriosclerotic disease.

Cytoplasmic estradiol binding sites in testis of fertile and infertile men.

Free cytoplasmic estradiol receptor was determined in testicular biopsies from six adults showing various primary testicular disorders, and in testicular tissue obtained at autopsy from 18 males of various ages. Estradiol receptors were found in all cases examined. Receptor concentration varied during testicular maturation and from one patient to another. The presence of the receptor at an early stage of development and the changes observed in its concentration during testicular maturation and dysfunction support the idea that estradiol may play, through its receptor interaction, an important role in the regulation of testicular steroidogenesis during postnatal human development.

Endocrine effects of vasectomy.

In order to provide information on the endocrine effects of vasectomy, unconjugated pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, oestrone and oestradiol were analysed in the blood plasma of twenty Mexican men on two occasions before and 1, 3, 6 and 12 months after vasectomy. Vasectomy appeared to be associated with a significant decrease in the plasma levels of pregnenolone, dehydroepiandrosterone and androstenedione and a significant increase in the levels of dihydrotestosterone and oestrone. A probably significant increase in oestradiol levels took place 12 months after vasectomy but not before. No consistent changes were found in testosterone (up to 12 months) or in FSH and LH levels (up to 6 months) after vasectomy. The unconjugated steroids indicated above, except oestrone, were also estimated, whenever possible, in seminal plasma specimens obtained from thirty-nine subjects (including the twenty indicated above) on the same occasions. Vasectomy was associated with a highly significant decrease of seminal plasma dihydrotestosterone levels on all occasions and a significant decrease in androstenedione levels after 6 and 12 months. After 12 months there was a decrease in dehydroepiandrosterone and an increase in oestradiol; these changes were both probably significant. In another preliminary study, the levels of pregnenolone sulphate, dehydroepiandrosterone sulphate, testosterone glucuronide, testosterone sulphate and dihydrotestosterone sulphate were estimated before and 1 month after vasectomy in the seminal plasma of fourteen to seventeen subjects. Testosterone glucuronide fell, probably significantly, but other conjugates were unchanged. The data indicate that vasectomy may be associated with significant changes in the circulating and in seminal plasma levels of several steroids. The gradual nature of some of the changes observed suggests the necessity of conducting in several centres large-scale, long-term studies on vasectomized subjects and on a carefully matched control group. During the last decade vasectomy has been widely practised in several parts of the world as a method of fertility control. However, information on the endocrine effects of this intervention appears to be scanty. In most of the human studies reported, a small number of individuals were investigated and the studies have been confined to the assessment of the short-term effects of the operation. Moreover, the hormonal indices assessed by the various investigators have been limited, in most cases, to gonadotrophins and testosterone in blood. The present study was designed to assess in the same subjects the levels of a number of unconjugated steroids, FSH and LH on two occasions before and 1, 3, 6 and 12 months after vasectomy. The studies were extended to include steroid analyses in seminal plasma in the hope that such assays might yield information as to the effects of vasectomy on the distribution of steroids in the fluids of the male reproductive tract.

Reduction of the oral contraceptive estrogen burden by alternate day estrogen administration.

PIP: Reduction of the oral contraceptive estrogen burden by alternate-day estrogen administration was studied. 3 regimens: 1) 1 mg norethindrone acetate (NET-Ac) plus .05 mg ethinyl estradiol (EE) on alternate days, 2) .05 mg NET plus .03 mg EE daily, and 3) .05 mg NET daily plus .06 EE on alternate days, were compared. Studies with the 1st regimen were prematurely terminated due to gross cycle irregularities; the 2nd regimen provided better cycle control but inadequate pregnancy protection apparently because of inconsistent inhibition of ovulation. Studies with the last regimen, expanded to include 1090 women for 12,942 patient-months, had clinically acceptable bleeding patterns with a bleeding discontinuation rate after the 1st year of 10.5 and after the 2nd year of 11.9. 2 of 8 pregnancies which occurred were attributed to method failure.^ieng