Usefulness of open data to determine the incidence of COVID-19 and its relationship with atmospheric variables in Spain during the 2020 lockdown.

The SARS-CoV-2 pandemic and the spread of the COVID-19 disease led to a lockdown being imposed in Spain to minimise contagion from 16 March 2020 to 1 May 2020. Over this period, measures were taken to reduce population mobility (a key factor in disease transmission). The scenario thus created enabled us to examine the impact of factors other than mobility (in this case, meteorological conditions) on the incidence of the disease, and thus to identify which environmental variables played the biggest role in the pandemic's evolution. Worthy of note, the data required to perform the study was entirely extracted from governmental open data sources. The present work therefore demonstrates the utility of such data to conduct scientific research of interest to society, leading to studies that are also fully reproducible. The results revealed a relationship between temperatures and the spread of COVID-19. The trend was that of a slightly lower disease incidence as the minimum temperature rises, i.e. the lower the minimum temperature, the greater the number of cases. Furthermore, a link was found between the incidence of the disease and other variables, such as altitude and proximity to the sea. There were no indications, however, in the study's data, of a relationship between incidence and precipitation or wind.

Toxoplasma gondii infection in hospitalized people living with HIV in Spain, 1997 to 2015.

Toxoplasma gondii infection was one of the most frequent AIDS-defining conditions in HIV-infected individuals until the advent of combination antiretroviral therapy. We aimed to assess the clinical load, coinfection, and mortality, as well as time trends for people living with HIV and hospitalized with Toxoplasma gondii infection, in Spain from 1997 to 2015. Retrospective observational analysis using the Spanish National Registry of Hospital Discharges. Information was retrieved for the study period using the International Classification of Diseases, 9th revision. There were 66,451,094 hospital admissions in Spain from 1997 to 2015, including 472,269 (0.71%) in people living with HIV. Toxoplasma gondii infection was registered in 9006 of these (overall prevalence 1.91%), making it the fifth most common opportunistic infection in hospitalized HIV-positive patients. Prevalence of Toxoplasma gondii infection declined in this group from 4.2% in 1997 to 0.8% in 2015 (p < 0.001), while mean age increased, from 35 years in 1997 to 44 years in 2015. The overall in-hospital mortality rate declined from 13.5% in 1997 to 8.9% in 2015, and it was higher in the concomitant presence of bacterial pneumonia (28.9% vs. 10.2%, p < 0.001), cryptosporidiosis (26.9% vs. 11.5%; p = 0.03), cytomegalovirus disease (18.2% vs. 11.2%, p < 0.001), Pneumocystis jiroveci pneumonia (31.5% vs. 10.5%, p < 0.001), leukoencephalopathy (19.8% vs. 11.78% p < 0.001), and wasting syndrome (29.3% vs 10.9%; p < 0.001). Toxoplasma gondii infection prevalence has significantly declined among hospitalized HIV-infected patients in Spain during the last two decades, coinciding with the widespread use of combination antiretroviral therapy.

Screening ultrafiltration membranes to separate lactose and protein from sheep whey: application of simplified model.

Several studies demonstrated that protein from whey milk could be a new strategy to reduce energy intake and increase satiety. Sheep whey has high protein content, but it is also rich in lactose. The aim of this study was to screening different ultrafiltration membranes to separate protein and lactose from sheep whey in one step. Protein was recovered in the concentrate feed, and lactose passed through three membranes and was recovered in the permeate feed. Membranes with different chemical composition and molecular weight cut-offs were assayed, and the influence of operating pressure and lactose concentration feed in the permeate flux and lactose rejection coefficients were studied. Lactose separation was not affected by pressure in GR60PP or GR90PP, and 85% and 80%, respectively of the lactose was separated into permeate feed. When the feed concentration increased, lactose separation remained stable in all three membranes, being GR60PP the most efficient, as 90% of the disaccharides were separated. In all cases 100% of the protein was recovered. Finally, the Spiegler-Kedem-Katchalsky model perfectly fitted the results obtained about lactose rejection coefficients.

Ultrafiltration Membranes Modified with Reduced Graphene Oxide: Effect on Methyl Green Removal from Aqueous Solution.

In this work, three types of ultrafiltration membranes with different characteristics (GR60PP, RC70PP and GR80PP) have been tested for the removal of the dye methyl green. The tests were first carried out with the three membranes without any modification and then with the membranes' surfaces modified with reduced graphene oxide (rGO). The modification was achieved through physical treatment. The CR70PP membrane did not support the modification treatment and was discarded. The other membranes were initially characterized with distilled water tests to study the permeability to the solvent, and later, the permeate fluxes and the values of rejection coefficients were obtained at different working pressures with a fixed dye initial concentration. In addition, SEM images and SEM-EDX spectra of the native and modified membranes were obtained before and after the dye tests. The GR60PP membrane has shown the best results in relation to the modification because it has increased its rejection levels. On the opposite, the GR80PP membrane performs better without surface modification, achieving the highest rejection values and the highest permeate fluxes in its native form.

Prediction of Flux and Rejection Coefficients in the Removal of Emerging Pollutants Using a Nanofiltration Membrane.

The removal of three emerging pollutants: carbamazepine, ketoprofen, and bisphenol A, has been studied using the nanofiltration flat sheet membrane NF99HF. The removal efficiencies of the membrane have been evaluated by two system characteristic parameters: permeate flux and rejection coefficient. The influence of two operating variables has been analysed: operating pressure and feed concentration. Before and after the tests with emerging pollutants, the membrane has been characterized by determining its water permeability coefficient and its magnesium chloride rejection coefficient to find out if the removal of emerging pollutants causes membrane fouling. The results show that operating pressure has significant separation effects, obtaining the highest efficiencies at a pressure of 20 bar for pollutant concentrations between 5 and 25 mg/L. Moreover, rejection of ketoprofen was found to be dependent on electrostatic repulsion, while rejection of bisphenol A was significantly affected by adsorption onto the membrane. Finally, the experimental data have been fitted to the solution diffusion model and to the simplified model of Spiegler-Kedem-Katchalsky to predict the behaviour of the nanofiltration membrane in the removal of the tested pollutants. Good agreement between the experimental and predicted carbamazepine and bisphenol A data has been obtained with each model, respectively.

Ibuprofen Removal by Graphene Oxide and Reduced Graphene Oxide Coated Polysulfone Nanofiltration Membranes.

The presence of pharmaceutical products, and their metabolites, in wastewater has become a focus of growing environmental concern. Among these pharmaceutical products, ibuprofen (IBU) is one of the most consumed non-steroidal anti-inflammatory drugs and it can enter the environment though both human and animal consumption, because it is not entirely absorbed by the body, and the pharmaceutical industry wastewater. Nanofiltration has been described as an attractive process for the treatment of wastewater containing pharmaceutical products. In this paper, the modification of a polysulfone nanofiltration membrane by coating with graphene oxide (GO) and reduced graphene oxide (RGO) has been carried out. The morphology and elemental composition of the active layer of unmodified and modified membranes were analyzed by scanning electronic microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX), respectively. Initial characterization membranes was carried out, studying their water permeability coefficient and their permeate flux and rejection coefficients, at different applied pressures, using magnesium chloride solutions. The behavior of both pristine and coated membranes against ibuprofen solutions were analyzed by studying the permeate fluxes and the rejection coefficients at different pressures and at different contaminant concentrations. The results have shown that both GO and RGO coated membranes lead to higher values of ibuprofene rejection than that of uncoated membrane, the latter being the one that presents better results in the studies of permeability, selectivity, and fouling.

Fluoride rearrangement reactions of polyphenyl- and polyvinylsilsesquioxanes as a facile route to mixed functional phenyl, vinyl T10 and T12 silsesquioxanes.

Polyphenylsilsesquioxane [PhSiO(1.5)](n) (PPS) and polyvinylsilsesquioxane [vinylSiO(1.5)](n) (PVS) are polymeric byproducts of the syntheses of the related T(8) octamers [PhSiO(1.5)](8) and [vinylSiO(1.5)](8). Here we demonstrate that random-structured PPS and PVS rearrange in the presence of catalytic amounts of Bu(4)N(+)F(-) in THF to form mixed-functionality polyhedral T(10) and T(12) silsesquioxane (SQ) cages in 80-90% yields. Through control of the initial ratio of starting materials, we can statistically tailor the average values for x for the vinyl(x)Ph(10-x)T(10) and vinyl(x)Ph(12-x)T(12) products. Metathetical coupling of x approximately = 2 vinyl cages with 4-bromostyrene produces SQs with an average of two 4-bromostyrenyl substituents. These products can be reacted via Heck coupling with vinylSi(OEt)(3) to produce SQs with vinylSi(OEt)(3) end-caps. Alternately, Heck coupling with the originally produced x approximately = 2 vinyl SQs leads to "beads on a chain" SQ oligomers joined by conjugated organic tethers. The functionalized T(10) and T(12) cages, metathesis, and Heck compounds were characterized by standard analytical methods (MALDI-TOF MS, (1)H and (13)C NMR spectroscopy, TGA, and GPC). MALDI confirms the elaboration of the cages after each synthetic step, and GPC verifies the presence of higher molecular weight SQ oligomers. TGA shows that all of these compounds are thermally stable in air (>300 degrees C). The UV-vis absorption and emission behavior of the Heck oligomers reveals exceptional red-shifts (> or = 60 nm) compared to the vinylSi(OEt)(3) end-capped model compounds, suggesting electronic interactions through the SQ silica cores. Such phenomena may imply 3-D conjugation through the cores themselves.

Synthesis and photophysical properties of stilbeneoctasilsesquioxanes. Emission behavior coupled with theoretical modeling studies suggest a 3-D excited state involving the silica core.

A set of stilbene-substituted octasilicates [p-RStil(x)Ph(8-x)SiO(1.5)](8) (R = H, Me, MeO, Cl, NMe(2) and x = 5.3-8) and [o-MeStilSiO(1.5)](8) were prepared. Model compounds were also prepared including the corner and half cages: [p-MeStilSi(OEt)(3)], [p-Me(2)NStilSi(OSiMe(3))(3)], and [p-Me(2)NStilSi(O)(OSiMe)](4). These compounds were characterized by MALDI-TOF, TGA, FTIR, and (1)H NMR techniques. Their photophysical properties were characterized by UV-vis, two-photon absorption, and cathodoluminescence spectroscopy (on solid powders), including studies on the effects of solvent polarity and changes in concentration. These molecules are typically soluble, easily purified, and robust, showing T(d(5%)) > 400 degrees C in air. The full and partial cages all show UV-vis absorption spectra (in THF) identical to the spectrum of trans-stilbene, except for [o-MeStilSiO(1.5)](8), which exhibits an absorption spectrum blue-shifted from trans-stilbene. However, the partial cages show emissions that are red-shifted by approximately 20 nm, as found for stilbene-siloxane macrocycles, suggesting some interaction of the silicon center(s) with the stilbene pi* orbital in both the corner and half cages. In contrast, the emission spectra of the full cages show red-shifts of 60-100 nm. These large red-shifts are supported by density functional theoretical calculations and proposed to result from interactions of the stilbene pi* orbitals with a LUMO centered within the cage that has 4A(1) symmetry and involves contributions from all Si and oxygen atoms and the organic substituents. Given that this LUMO has 3-D symmetry, it appears that all of the stilbene units interact in the excited state, consistent with theoretical results, which show an increased red-shift with an increase in the functionalization of a single corner to functionalization of all eight corners with stilbene. In the case of the Me(2)N- derivatives, this interaction is primarily a charge-transfer interaction, as witnessed by the influence of solvent polarity on the emission behavior. More importantly, the two-photon absorption behavior is 2-3 times greater on a per p-Me(2)Nstilbene basis for the full cage than for the corner or half cages. Similar observations were made for p-NH(2)stilbenevinyl(8)OS cages, where the greater conjugation lengths led to even greater red-shifts (120 nm) and two-photon absorption cross sections. Cathodoluminescence studies done on [p-MeStilSiO(1.5)](8) or [p-MeStilOS](8) powders exhibit essentially the same emissions as seen in solution at high dilution. Given that only the emissions are greatly red-shifted in these molecules, whereas the ground-state UV-vis absorptions are not changed from trans-stilbene, except for the ortho derivative, which is blue-shifted 10 nm. It appears that the interactions are only in the excited state. Theoretical results show that the HOMO and LUMO states are always the pi and pi* states on the stilbene, which show very weak shifts with increasing degrees of functionalization, consistent with the small changes in the UV-vis spectra. The band gap between the lowest unoccupied 4a1 symmetry core state localized inside the silsesquioxane cage and the highest occupied state (pi state on stilbene), however, is markedly decreased as the number of stilbene functional groups is increased. This is consistent with the significant red-shifts in the emission spectra. The results suggest that the emission occurs from the 4a1 state localized on the cage. Moreover, for the compounds [p-RStil(6-7)Ph(2-1)OS](8), the emissions are blue-shifted compared to those of the fully substituted compounds, suggesting the molecular symmetry is reduced (from cubic), thereby reducing the potential for 3-D delocalization and raising the energy of the LUMO. The implications are that these octafunctional molecules exhibit some form of 3-D interaction in the excited state that might permit their use as molecular transistors as well as for energy collection and dispersion as molecular antennas, for example, and for nonlinear optical applications.

Adding access blood flow surveillance reduces thrombosis and improves arteriovenous fistula patency: a randomized controlled trial.

PURPOSE: Stenosis is the main cause of arteriovenous fistula (AVF) failure. It is still unclear whether surveillance based on vascular access blood flow (QA) enhances AVF function and longevity. METHODS: We conducted a three-year follow-up randomized, controlled, multicenter, open-label trial to compare QA-based surveillance and pre-emptive repair of subclinical stenosis with standard monitoring/surveillance techniques in prevalent mature AVFs. AVFs were randomized to either the control group (surveillance based on classic alarm criteria; n = 104) or to the QA group (QA measured quarterly using Doppler ultrasound [M-Turbo®] and ultrasound dilution [Transonic®] added to classic surveillance; n = 103).The criteria for intervention in the QA group were: 25% reduction in QA, QA<500 mL/min or significant stenosis with hemodynamic repercussion (peak systolic velocity [PSV] more than 400 cm/sc or PSV pre-stenosis/stenosis higher than 3). RESULTS: At the end of follow-up we observed a significant reduction in the thrombosis rate in the QA group (0.025 thrombosis/patient/year in the QA group vs. 0.086 thrombosis/patient/year in the control group [p = 0.007]). There was a significant improvement in the thrombosis-free patency rate (HR, 0.30; 95% CI, 0.11-0.82; p = 0.011) and in the secondary patency rate in the QA group (HR, 0.49; 95% CI, 0.26-0.93; p = 0.030), with no differences in the primary patency rate between the groups (HR, 0.98; 95% CI, 0.57-1.61; p = 0.935).There was greater need for a central venous catheter and more hospitalizations associated with vascular access in the control group (p = 0.034/p = 0.029).Total vascular access-related costs were higher in the control group (€227.194 vs. €133.807; p = 0.029). CONCLUSIONS: QA-based surveillance combining Doppler ultrasound and ultrasound dilution reduces the frequency of thrombosis, is cost effective, and improves thrombosis free and secondary patency in autologous AVF.

The impact of access blood flow surveillance on reduction of thrombosis in native arteriovenous fistula: a randomized clinical trial.

PURPOSE: The usefulness of access blood flow (QA) measurement is an ongoing controversy. Although all vascular access (VA) clinical guidelines recommend monitoring and surveillance protocols to prevent VA thrombosis, randomized clinical trials (RCTs) have failed to consistently show the benefits of QA-based surveillance protocols. We present a 3-year follow-up multicenter, prospective, open-label, controlled RCT, to evaluate the usefulness of QA measurement using Doppler ultrasound (DU) and ultrasound dilution method (UDM), in a prevalent hemodialysis population with native arteriovenous fistula (AVF). METHODS: Classical monitoring and surveillance methods are applied in all patients, the control group (n = 98) and the QA group (n = 98). Besides this, DU and UDM are performed in the QA group every three months. When QA is under 500 ml/min or there is a >25% decrease in QA the patient goes for fistulography, surgery or close clinical/surveillance observation. Thrombosis rate, assisted primary patency rate, primary patency rate and secondary patency rate are measured. RESULTS: After one-year follow-up we found a significant reduction in thrombosis rate (0.022 thrombosis/patient/year at risk in the QA group compared to 0.099 thrombosis/patient/year at risk in the control group [p = 0.030]). Assisted primary patency rate was significantly higher in the QA group than in control AVF (hazard ratio [HR] 0.23, 95% confidence interval [CI] 0.05-0.99; p = 0.030). In the QA group, the numbers unddergoing angioplasty and surgery were higher but with no significant difference in non-assisted primary patency rate (HR 1.41, 95% CI 0.72-2.84; p = 0.293). There was a non-significant improvement in secondary patency rate in the QA group (HR 0.510, 95% CI 0.17-1.50; p = 0.207). CONCLUSIONS: The measurement of QA combining DU and UDM shows a reduction in thrombosis rate and an increased assisted primary patency rate in AVF after one-year follow-up. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02111655.

Effect of diabetes mellitus on Corvis ST measurement process.

PURPOSE: Diabetes mellitus (DM) affects corneal biomechanical parameters. We compared analyses using ORA (Ocular response analyser) and Corvis ST to determine the influence of disease duration, hyperglycaemia and haemoglobin A1c (HbA1c) levels on these parameters. METHODS: This observational, cross-sectional, observer-masked study assessed one eye of 94 consecutive DM patients and 41 healthy subjects. Two DM groups were analysed: the uncontrolled DM group (n = 54) (HbA1c ≥ 7%) and the controlled DM group (n = 40) (HbA1c < 7%). Central corneal thickness (CCT) was measured by ultrasonic pachymetry and intraocular pressure (IOP) by Goldmann applanation tonometry. ORA and Corvis ST analyses were performed to evaluate the changes. RESULTS: Most of the Corvis ST parameters [Deformation amplitude (DA), A1 and A2 times, A1 velocity] in the uncontrolled DM group eyes were found to be significantly different to controls and controlled DM group eyes (p = 0.005, p = 0.001, p < 0.0001, p = 0.002, respectively). DA on the Corvis ST was correlated with blood glucose concentration (p = 0.004) and HbA1c percentage (p = 0.002). ORA corneal hysteresis was significantly lower in diabetic patients with elevated HbA1c than in control subjects (p = 0.001) and was affected by disease duration (p = 0.037), whereas the corneal resistance factor remained unaltered. CONCLUSIONS: A poor glucose control in DM affects corneal biomechanics measured by ORA and Corvis ST, which may cause high IOP measurements independent of CCT. The measurement of the corneal biomechanics should be taken into consideration in the clinical practice.

Displacement of T4 from transthyretin by the synthetic flavonoid EMD 21388 results in increased production of T3 from T4 in rat dams and fetuses.

EMD 21388 displaces T4 from circulating transthyretin, is a potent in vitro inhibitor of outer-ring deiodination (5'D) of T4 and affects thyroid hormone secretion. To study its extrathyroidal effects on the thyroid hormone status of pregnant dams and their fetuses, we treated the dams with methimazole and infused them with T4 and with either 2.5 mg EMD 21388/rat per day [(EMD(+)], or placebo solution [EMD(-)]. EMD reduced total T4 and T3 in the maternal circulation, but free T4 increased and free T3 decreased. The total amount of T3 generated from T4 in the maternal compartment increased. Placental T3 also increased in EMD(+) animals, T4 remaining the same. EMD also reached the fetal circulation. The total fetal extrathyroidal T4 pool decreased to half that of EMD(-) fetuses, whereas T3 increased 1.8-fold, thus mitigating fetal T3 deficiency, especially in the lung. Thus, if the maternal supply of T4 is kept constant, EMD mitigates the T3 deficiency of many tissues of the hypothyroid fetus. Most of the effects of this dose of EMD could result from the displacement of T4 from circulating transthyretin. Liver 5'D-I activity did not decrease, but actually increased by 40% in dams and fetuses. The enhanced transfer of T4 into tissues would also increase the amount of substrate available to 5'D-I, leading to an increased amount of T3 in maternal and fetal pools. This had not been anticipated from the changes in circulating T4 and T3, whether maternal or fetal, total or free.

The role of the CAG repeat polymorphism in the androgen receptor gene and of skewed X-chromosome inactivation, in the pathogenesis of hirsutism.

The human androgen receptor (AR) gene contains a variable number of CAG repeats within exon 1. Shorter AR alleles, by increasing transactivation, may result in augmented AR-mediated sensitivity of the hair follicle. We have evaluated whether the number of CAG repeats, or if skewed inactivation of X-chromosome, favoring the presence of shorter AR alleles, influence hirsutism in women with and without hyperandrogenemia. Twenty-eight women with idiopathic hirsutism (normal serum androgens), 34 women with hyperandrogenic hirsutism (increased serum androgens), and 15 healthy control women were analyzed by evaluating the number of CAG repeats in genomic DNA, as well as the methylation pattern (after DNA digestion with HpaII), which allows identification of which allele is inactive. Despite marked differences among the groups in serum androgens, which were markedly increased in women with hyperandrogenic hirsutism as compared with women with idiopathic hirsutism and to controls, there were no significant differences among the groups in the number of CAG repeats. Moreover, skewed X-chromosome inactivation was found in 10 subjects (3 with idiopathic hirsutism, 5 with hyperandrogenic hirsutism, and 2 controls; P = 0.746) of the 67 women (14.9%) who were heterozygous for the AR gene. In several of these subjects, it was the shorter allele that was preferentially inactivated. In conclusion, neither the CAG repeat polymorphism in the AR gene, nor skewed X-chromosome inactivation, seem to play a significant role in the pathogenesis of hirsutism.

Increase in plasma thyrotropin levels in hypothyroid patients during treatment due to a defect in the commercial preparation.

Around mid-1995, the Molecular Endocrinology Laboratory of the Regional Hospital (Malaga, Spain) began detecting an increase in TSH levels in the serum of patients under study to control the treatment of hypothyroidism with levothyroxine. Over a period of 5 months, of a total of 467 hypothyroid patients treated with Levothyroid, 53% had TSH levels higher than 6 microU/mL. The reliability of the biochemical results was verified by duplicating 56 randomly chosen samples from all those with high TSH levels and by an external control performed in four different laboratories. The amount of levothyroxine in the tablets was analyzed by RIA, high performance liquid chromatography, and their iodine contents. The lowest levels of levothyroxine found in the 50 micrograms Levothyroid tablets were those determined by RIA, with a mean value of 32.3 micrograms, resulting in a 35.3% loss of activity. The mean value of levothyroxine found in these same tablets by high performance liquid chromatography was 39.3 micrograms, amounting to a 21.3% loss in activity. The iodine showed no significant loss in these tablets, with a mean experimental value of 48 micrograms. The commercial laboratory withdrew lot J from the market, the one in which these deficiencies were found.

A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain.

We prospectively estimated the prevalence of the polycystic ovary syndrome (PCOS), as defined by the NIH/NICHHD 1990 endocrine criteria, in a population of 154 Caucasian women of reproductive age reporting spontaneously for blood donation. Anthropometric data; the presence of hirsutism, acne, and androgenic alopecia; and the menstrual history were recorded by a single investigator. In 145 women, blood samples were also obtained for measurement of serum androgen levels. PCOS was defined by the presence of 1) oligomenorrhea, 2) clinical and/or biochemical hyperandrogenism, and 3) exclusion of hyperprolactinemia, thyroid disorders, and nonclassic 21-hydroxylase deficiency. Hirsutism was defined by a modified Ferriman-Gallwey score of 8 or more, acne was considered as a sign of hyperandrogenism when persistent after the second decade of life, and hyperandrogenemia was defined by an increase in circulating testosterone or dehydroepiandrosterone sulfate or an increase in the free androgen index above the 95th percentile of the control values derived from the nonhirsute, nonacneic women having regular menses who were not receiving hormonal therapy. PCOS was present in 10(6.5%), hirsutism was present in 11 (7.1%), and acne was present in 19 (12.3%) of the 154 women. Our results demonstrate a 6.5% prevalence of PCOS, as defined, in a minimally biased population of Caucasian women from Spain. The polycystic ovary syndrome, hirsutism, and acne are common endocrine disorders in women.

Screening for mutations in the steroidogenic acute regulatory protein and steroidogenic factor-1 genes, and in CYP11A and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome, gene-1 (DAX-1), in hyperandrogenic hirsute women.

Abstract Abnormalities in adrenal and/or ovarian steroidogenesis are found in most patients with hirsutism. The rate-limiting step in the synthesis of steroids in the ovary and the adrenal is the conversion of cholesterol into pregnenolone by cholesterol side-chain cleavage enzyme (P450scc), encoded by the gene CYP11A, after cholesterol is introduced into the mitochondria by the steroidogenic acute regulatory protein (StAR). DAX-1 is a repressor of StAR gene expression, and steroidogenic factor-1 (SF-1) is a regulator of CYP11A, DAX-1, and StAR gene. Mutations in any of these factors resulting in gain of function, or loss of repression, of StAR or P450scc might contribute to the steroidogenic abnormalities present in hirsute patients. In the present study we have screened, using heteroduplex analysis, the genes encoding StAR and SF-1 as well as DAX-1 and CYP11A for mutations in genomic DNA from 19 women presenting with hirsutism and increased serum androgen levels. When variants were found, analysis was extended to a larger group of hyperandrogenic patients and nonaffected women. Two variants were identified in the SF-1 gene. A G-->C change in exon 6, resulting in an Arg(365)Pro mutation, was found in 1 of 45 patients, but not in controls. Also, a Gly(146)Ala missense mutation, resulting from a G-->C change in exon 4, was found in 2 of 48 patients and in 2 of 50 nonaffected individuals. We identified a C-->T base pair change at position -33 of the StAR gene. Three of 48 patients and 3 of 43 controls presented this variant. No mutations were found in coding regions of the StAR gene. Analysis of CYP11A-coding regions identified a G-->A change in exon 3, resulting in a Val(179)Ile missense mutation. This mutation was found in 1 of 29 patients studied and was not present in 50 controls. Finally, analysis of DAX-1 showed no variant in any of the women studied. In conclusion, mutations in StAR, SF-1, CYP11A, and DAX-1 are seldom found in hirsute patients and do not explain the steroidogenic abnormalities found in these women.

Analysis of the 5' non-coding region of rat liver S-adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme.

A 3 kb cDNA coding for rat liver S-adenosylmethionine (AdoMet) synthetase has been isolated. The Mr of the protein has been unequivocally determined by cDNA sequencing and enzyme purification on a thiopropyl-Sepharose column. The length of the mRNA 5' non-coding region has been defined by primer-extension analysis. The rat liver cloned cDNA has been also used to detect S-adenosylmethionine synthetase mRNA in human liver.

Improvement in HPLC fractionation of thyroxine-containing thyroglobulin tryptic peptides by prior Accell ion-exchange column chromatography.

The isolation and purification of the peptides containing the hormonogenic tyrosyl residues in thyroglobulin is of great interest to the understanding of structure-function relationships in this iodoprotein. This is usually performed in reduced alkylated selectively hydrolyzed thyroglobulin by subsequent HPLC fractionation. However, the main difficulty encountered when trying to isolate these peptides is their disproportion with respect to the total number of possible peptides (14 vs a total of 508). Several HPLC runs with different mobile phases are necessary and each run is accompanied with significant losses of the peptides to be purified. In an attempt to improve the separation of the T4-containing peptides in thyroglobulin tryptic digests from the much more abundant iodotyrosine-containing ones, which are present as contaminants, we have used a very simple and fast step prior to the HPLC fractionation as it is a self-packed ion-exchange column chromatography. This preliminary step results in an improvement in the separation of these peptides and leads to a relative enrichment of the hormonogenic peptides falling in different zones of the HPLC chromatogram, which facilitates their subsequent separation and purification by HPLC.

Efficient thyroid hormone formation by in vitro iodination of a segment of rat thyroglobulin fused to Staphylococcal protein A.

A polypeptide of 224 amino acids from the C terminus of rat thyroglobulin fused to Staphylococcal protein A (TgC 224), containing 3 tyrosines which have been shown to be hormonogenic in vivo (Tyr-2555, -2569 and -2748), forms thyroid hormones with relatively high efficiency upon in vitro enzymatic iodination using, most likely, the hormonogenic Tyr-2555 and Tyr-2569. Acetylcholinesterase, which has sequence and structural homology with the C terminus of the thyroglobulin molecule and bovine serum albumin, used as control proteins, formed thyroid hormones with lower efficiency. These results validate our experimental approach to define the structural requirements for thyroid hormone formation using thyroglobulin fragments.

Receiver operating characteristic analysis of the performance of basal serum hormone profiles for the diagnosis of polycystic ovary syndrome in epidemiological studies.

OBJECTIVE: We have used receiver operating characteristic (ROC) analysis to determine the diagnostic performance of several serum parameters, in order to evaluate their potential usefulness in establishing the diagnosis of polycystic ovary syndrome (PCOS) in epidemiological studies. DESIGN: Prospective study. METHODS: One hundred and fourteen women reporting spontaneously for blood donation were included in the study. Menopausal and oral contraceptive-treated women were excluded. Serum samples were obtained at the moment of donation, independently of fasting, time of day or day of menstrual cycle. Measurements included total testosterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), LH, FSH and estradiol. The free testosterone (FT) concentration and the free androgen index (FAI) were calculated from testosterone and SHBG levels. ROC curves were calculated for all these serum determinations. RESULTS: Eight patients were diagnosed with PCOS, according to the presence of oligomenorrhea, hirsutism, acne and/or hyperandrogenemia, and exclusion of non-classic congenital adrenal hyperplasia, hypothyroidism and hyperprolactinemia. Of the parameters studied SHBG, FAI, FT and DHEAS were considered adequate measures for the diagnosis of PCOS. For example, serum SHBG levels showed an area under the ROC curve of 0.875+/-(S.E.(w))0.045 (95% confidence interval 0.800-0.929). A SHBG decision threshold <37 nmol/l had a sensitivity of 87.5%, a specificity of 86.8%, a positive likelihood ratio of 6.63, and a negative likelihood ratio of 0.14, for the diagnosis of PCOS. CONCLUSIONS: Our present results strongly suggest that decreased SHBG levels, and increased FAI, free testosterone concentration and DHEAS concentrations, are highly effective as single analytical procedures in epidemiological studies for the detection of PCOS in women of reproductive age.