Severe endometriosis: low value of AMH did not affect oocyte quality and pregnancy outcome in IVF patients.

OBJECTIVE: The aim of this pilot study was to determine whether the low anti-müllerian hormone (AMH) serum level, due to severe endometriosis, was associated with diminished oocyte yield, poor oocyte/embryo quality and reduced in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) clinical outcomes in young patients (<37 years old). PATIENTS AND METHODS: A total of 50 IVF cycles of patients younger than 37 with severe endometriosis were retrospectively analyzed in a single center between November 2016 and July 2018. The clinical outcome was then compared to a control group of 84 patients with no story of endometriosis and normal AMH value. AMH value was evaluated within three months before the stimulation. In these two groups, number and maturation of retrieved oocytes, embryo quality, and pregnancy outcomes were evaluated and compared using Student's t-test and Fisher's test. RESULTS: The number of oocytes retrieved per cycle and the percentage of mature oocytes (MII) were significantly lower (p < 0.001) in IVF patients with severe endometriosis and AMH value ≤ 1.1 ng/ml (Group A; 3.8±2.6 retrieved oocytes, 70% MII) compared to patients without endometriosis and AMH levels > 1.1 ng/ml (Group B; 6.9±4.6 retrieved oocytes, 83% MII). On the other hand, embryo morphology, implantation rate (31% vs. 33%; p = 0.833) and pregnancy rate (50% vs. 49%; p = 1) were comparable in the two groups. CONCLUSIONS: This study shows that younger patients with an impairment of the ovarian reserve due to severe endometriosis, displayed a diminished oocyte yield but not a reduction in embryo quality and pregnancy outcomes. These results suggest that serum AMH levels should not be adopted as a criterion for discouraging these patients from undergoing IVF/ICSI treatments.

Pro-Inflammatory Cytokines in Nasopharyngeal Aspirate From Hospitalized Children With Respiratory Syncytial Virus Infection With or Without Rhinovirus Bronchiolitis, and Use of the Cytokines as Predictors of Illness Severity.

Respiratory syncytial virus (RSV) and human rhinovirus (HRV) respiratory infection in children induce production of inflammatory interleukins (ILs) in the respiratory epithelium. As IL(s) determine the severity of illness, the purpose of this study was to identify the pro-inflammatory IL(s) that could be predictor(s) of clinical severity. One hundred and fifteen patients <2 years old with bronchiolitis due to RSV and /or HRV and 38 controls were selected from a hospital and an outpatient clinic. Clinical data of all patients were recorded. Severity was defined by the number of days with oxygen need. Nasopharyngeal aspirates (NPA) were collected to perform viral diagnosis by quantitative reverse transcription and polymerase chain reaction (qRT-PCR) and to quantify ILs: TNF-α, IL-10, IL-6, IL-1ß, and IL-8, by flow cytometry. Simple and multiple regression and receiver operating characteristic (ROC) curves were used for statistical analysis. Of the patients selected 60 were single RSV, 28 RSV associated to HRV, and 27 single HRV. All patients (115) showed significantly higher IL levels when compared with controls. Levels of IL-6, IL-1ß, and IL-8 detected in NPA from RSV single and associated to HRV were significantly higher than HRV infected and positively associated with days requiring O2.Levels of IL-6, IL-1ß, and IL-8 detected in NPA from patients infected with RSV only or with both RSV and HRV are increased, and any of those 3 cytokines may have a predictive value for the number of days with need of supplemental oxygen.

Desarrollo de un queso maduro con adición del cultivo probiótico lactobacillus paracasei subsp. paracasei LC-01 / Development of mature cheese with the adittion of the probiotic culture lactobacillus paracasei subsp. paracasei LC-01

The probiotic culture Lactobacillus paracasei subsp. paracasei LC-01 was incorporated to a mature cheese in order to obtain a functional product for a minimal reasonable period before its consumption. The ideal period of maturation for the cheese was determined by evaluating with consumers the acceptability of the flavor and texture of samples after 13.30 and 45 days of maturation to 12 ºC and 85 percent RH. The process of cheese elaboration was modified by pressing with a number of viable probiotic cells up to 1 x 106 UFC. A significant increment (alpha=5 percent) was detected in the re-counts of Lactobacillus paracasei subsp. paracasei for the three studied times of maturation. The pH value of the product fell throughout the 45 days of maturation. There were not significant differences (alpha=5 percent) in the flavor and texture acceptability of the cheeses put under different times of maturation. The probiotic population stayed stable (Ix 107 UFCI g average)for a 15 days maturated cheese, throughout 45 days of storage at 5ºC.

A un queso maduro se le incorporó el cultivo probiótico Lactobacillus paracasei subsp. paracasei LC-01 para obtener un producto funcional por un período mínimo razonable para su consumo. Se determinó un período ideal de maduración para el queso evaluando con consumidores la aceptabilidad del sabor y la textura de muestras de 13,30 y 45 días de maduración a 12 ºC y 85 por ciento HR. Se modificó el proceso de elaboración del queso para obtener un queso recién prensado con un número de células probióticas viables mayor a 1 x 106 UFC. Se encontró un incremento significativo (alfa=5 por ciento) en los recuentos del Lactobacillus paracasei subsp. paracasei para los tres tiempos de maduración estudiados con respecto al queso recién prensado. Los valores de pH del producto disminuyeron a través de los 45 días de maduración. No se hallaron diferencias significativas (alfa=5 por ciento) en la aceptabilidad del sabor y la textura para los quesos sometidos a los distintos tiempos de maduración. La población del probiótico se mantuvo estable (1 x 107 UFC/ g en promedio) para un queso madurado 15 días, a lo largo de 49 días de almacenamiento a 5ºC, sin que se haya registrado una tendencia de descenso o crecimiento significativa (alfa=5 por ciento).

Asistencia ventilatoria no invasiva en el paciente crónico: criterios de selección / Noninvasive ventilation in chronic patient: selection criteria

En el tratamiento de los pacientes con insuficiencia respiratoria crónica, ya sea esta por una condición progresiva o estable, se ha incorporado en los últimos años la Ventilación Mecánica no Invasiva (VNI) como una alternativa eficiente en el manejo domiciliario. Su uso protocolizado ha logrado mejorarla evolución y sobrevida de estos pacientes, disminuyendo la morbilidad relacionada a exacerbaciones y mejorando la calidad de vida del paciente y su familia. Esto ha requerido establecer criterios estrictos de selección y revisar las contraindicaciones según consideraciones técnicas y éticas. Estas decisiones tienen implicancias económicas, tecnológicas y en la organización de una red de apoyo en que participe en forma fluida todo el equipo de salud. Se mencionan las recomendaciones existentes parainiciar la VNI y los criterios de selección considerados en el Programa Chileno de Asistencia Ventilatoria no Invasiva en Domicilio.

Asma de difícil control / Difficult to control asthma

Algunos pacientes con asma no controlan sus síntomas respiratorios pesar del uso de corticoides inhalados con dosis mayores o iguales a 600 microgramos de Budesonida o su equivalente. En ellos se plantean interesantes preguntas que exigen repuestas concretas y rápidas. Siempre debe evaluarse la influencia del ambiente y la adherencia al tratamiento. Algunos factores genéticos raros, puedenasociarse a una pobre respuesta. Existen patrones inflamatorios celulares que su reconocimiento permite el inicio de terapias específicas; sin embargo lo primero es confirmar y asegurar el diagnóstico correcto.

Corticoides inhalados en pediatría: efectos adversos / Corticosteroids inhaled in pediatrics: adverse effects

Los efectos sistemicos potenciales de los corticoides inhalados son motivo de preocupación y se han hecho esfuerzos en producir esteroides inhalatorios (EL) que tengan el menor efecto sistemico con el mejor efecto antinflamatorio. Datos al respecto estan empezando a parecer pero aún falta investigación para poder sacar conclusiones seguras, sobretodo en lo que se refiere a los niños menores de 3 años.

[Identification of mutation in the gene cystic fibrosis transmembrane regulator (CFTR) in Chilean patients with cystic fibrosis]. / Identificación de mutaciones en el gen CFTR en pacientes chilenos con fibrosis quística.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene, that codes for a chloride channel located in the apical surface of epithelial cells. The main role of this protein is the regulation of chloride transport, and secondarily, of sodium and water to the extracellular space. More than 900 gene mutations have been described, and their relative frequency in different populations depends on their ethnic origin. AIM: To report the findings of Chilean patients with cystic fibrosis, in whom the presence of 20 common mutations was analyzed. PATIENTS AND METHODS: Fifty seven patients with established diagnosis or suspicion of CF were studied. The simultaneous identification of 20 mutations and the normal delta F508 allele was done using polymerase chain reactions with a commercial assay. RESULTS: Eight mutations were found. Fifty patients fulfilled diagnostic criteria proposed by the Consensus Panel of the CF Foundation and 66% of alleles were identified in this group. delta F508 mutation was found in 45%. We did not identify mutations in any of the remaining 7 patients. CONCLUSIONS: Our results suggest that the majority of undetected mutations are associated with atypical phenotypes or that some patients in this series could have other diseases. We recommend to include mutation analysis in the evaluation of Chilean patients with CF. It is useful to establish prognosis and genetic counselling.

Montelukast dose selection in children ages 2 to 5 years: comparison of population pharmacokinetics between children and adults.

Montelukast, a leukotriene receptor antagonist, has demonstrated efficacy and tolerability in the treatment of asthma in patients age 6 years and older. The purpose of this open, one-period, multicenter population pharmacokinetic study was to identify a chewable tablet (CT) dose of montelukast for administration to children ages 2 to 5 years with asthma, yielding a single-dose pharmacokinetic profile (area under the plasma concentration-time curve [AUC]) comparable to that of the 10 mg film-coated tablet (FCT) dose in adults. Because patient numbers were small and the volume of blood that could be collected from individual 2- to 5-year-old patients was limited, a population pharmacokinetic approach was used to estimate population AUC (AUCpop). The 4 mg CT dose of montelukast was well tolerated and yielded an AUCpop (2721 ng.h/mL) similar to that of the adult AUCpop (2595 ng.h/mL) observed after a 10 mg FCT dose. These results support the selection of a 4 mg once-daily CT dose of montelukast for future efficacy and safety studies in children ages 2 to 5 years with asthma.

Isolation of pigment-binding early light-inducible proteins from pea.

The early light-inducible proteins (ELIPs) in chloroplasts possess a high sequence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strategy: perfusion chromatography in combination with preparative isoelectric focussing. Two heterogeneous populations of ELIPs were obtained after chromatographic separation of solubilized thylakoid membranes using a weak anion exchange column. One of these populations contained ELIPs in a free form providing the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic analysis were performed. Absorption spectra and TLC revealed the presence of chlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 nm typical for chlorophyll a bound to the protein matrix. The action spectrum of the fluorescence emission measured at 674 nm showed several peaks originating mainly from chlorophyll a. It is proposed that ELIPs are transient chlorophyll-binding proteins not involved in light-harvesting but functioning as scavengers for chlorophyll molecules during turnover of pigment-binding proteins.

Synthesis and assembly of the D1 protein into photosystem II: processing of the C-terminus and identification of the initial assembly partners and complexes during photosystem II repair.

In previous studies [van Wijk, K. J., Bingsmark, S., Aro, E.-M., & Andersson, B. (1995) J. Biol. Chem. 270, 25685-25695; van Wijk, K. J., Andersson, B., & Aro, E.-M. (1996) J. Biol. Chem 271, 9627-9636], we have demonstrated that D1 protein synthesized in isolated chloroplasts and thylakoids is incorporated into the photosystem II (PSII) core complex. By pulse-chase experiments in these in vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII rc), and through additional assembly steps the PSII core complex was formed. In this study, we have analyzed this assembly process in more detail, with special emphasis on the initial events, through further purification and analysis of the assembly intermediates by nondenaturing Deriphat-PAGE and by flatbed isoelectric focusing. The D2 protein was found to be the dominant PSII reaction center protein initially associating with the new D1 protein. This strongly suggests that the D2 protein is the primary "receptor" or stabilizing component during or directly after synthesis of the D1 protein. After formation of the D1-D2 heterodimer, cyt b559 became attached, whereas the psbI gene product was assembled as a subsequent step, thereby forming a PSII reaction center complex. Subsequent formation of the PSII core occurred by binding of CP47 and then CP43 to the PSII rc. The rapid radiolabeling of a minor population of a PSII core subcomplex without CP43 indicated that an association of newly synthesized D1 protein with a preexisting complex consisting of D2/cyt b55q/psbI gene product/CP47 was possibly occurring, in parallel to the predominant sequential assembly pathway. The kinetics of synthesis and processing of the precursor D1 protein were followed in isolated chloroplasts and were compared with its incorporation into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.

Isolation of hydrophobic membrane proteins by perfusion chromatography--purification of photosystem II reaction centers from spinach chloroplasts.

Ion exchange perfusion chromatography has been introduced for the isolation of hydrophobic membrane protein complexes from thylakoid membranes of spinach chloroplasts. By using this chromatographic technique, previously shown to be useful for the rapid isolation of soluble proteins (Regnier, F. E. (1991) Nature 350, 634-635), we have been able to isolate oxygen evolving photosystem II core complexes and photosystem II reaction center particles. Pure reaction centers could be isolated from photosystem II core complexes after a chromatographic step requiring only 6.5 mm, which is a substantial improvement in comparisons with previous procedures. The entire preparation of photosystem II core complexes and reaction center II particles could be completed in less than 2 h. The use of perfusion chromatography, as a versatile method for the isolation of hydrophobic membrane proteins from photosynthetic membranes, as well as for other biological membranes, will be discussed.

Degradation of the light-stress protein is mediated by an ATP-independent, serine-type protease under low-light conditions.

Green plants respond to light stress by induction of the light-stress proteins (ELIPs). These proteins are stable as long as the light stress persists but are very rapidly degraded during subsequent low light conditions. Here we report that the degradation of ELIPs is mediated by an extrinsic, thylakoid-associated protease which is already present in the membranes during light stress conditions. Partial purification of the protease by perfusion chromatography indicates that this proteolytic activity may be represented by a protein with an apparent molecular mass of 65 kDa. The ELIP-directed protease is localized in the stroma lamellae of the thylakoid membranes and does not require ATP or additional stromal factors for proteolysis. The protease has an optimum activity at pH 7.5-9.5 and requires Mg2+ for its activity. The ELIP-degrading protease show an unusual temperature sensitivity and becomes reversibly inactivated at temperatures below 20 degree C and above 30 degree C. Studies with protease inhibitors indicate that this enzyme belongs to the serine class of proteases. The enhanced degradation of ELIP in isolated thylakoid membranes after addition of the ionophore nigericin suggests that a trans-thylakoid delta pH or changes in ionic strength may be involved in the mechanism of protease activation.

Rapid isolation of photosystem I chlorophyll-binding proteins by anion exchange perfusion chromatography.

With the new method of anion exchange perfusion chromatography we have devised an extremely rapid technique to subfractionate spinach Photosystem I into its chlorophyll a containing core complex and various components of the Photosystem I light-harvesting antenna (LHC I). The isolation time for the LHC I subcomplexes following solubilisation of native Photosystem I was reduced from 50 h using traditional density centrifugation procedures down to only 10-25 min by perfusion chromatography. Within this very short period of isolation, LHC I has been obtained as subfractions highly enriched in Lhca2+3 (LHC I-680) and Lhca1+4 (LHC I-730). Moreover, other highly enriched subfractions of LHC I such as Lhca2, Lhca3 and Lhca1+2+4 were obtained where the later two populations have not previously been obtained in a soluble form and without the use of SDS. These various subfractions of the LHC I antenna have been characterised by absorption spectroscopy, 77 K fluorescence-spectroscopy and SDS-PAGE demonstrating their identities, functional intactness and purity. Furthermore, the analyses located a chlorophyll b pool to preferentially transfer its excitation energy to the low energy F735 chromophore, and located specifically the origin of the 730 nm fluorescence to the Lhca4 component. It was also revealed that Lhca2 and Lhca3 have identical light-harvesting properties. The isolated Photosystem I core complex showed high electron transport capacity (1535 µmoles O2 mg Chl(-1) h(-1)) and low fluorescence yield (0.4%) demonstrating its high functional integrity. The very rapid isolation procedure based upon perfusion chromatography should in a significant way facilitate the subfractionation of Photosystem I proteins and thereby allow more accurate functional and structural studies of individual components.

Perfusion chromatography-a new procedure for very rapid isolation of integral photosynthetic membrane proteins.

The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.