Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.

Reactive oxygen species have a great impact on spermatozoa function. Gametes from sole males born in captivity (F1) display lower quality than those from wild individuals. In this paper, the percentage of cells positive for dichlorofluorescein (DCF(+)) was determined by flow cytometry in wild and F1 animals, the effect of cryopreservation on DCF(+) cells was evaluated in both groups and the distribution of H2O2 within the cell was studied by confocal microscopy. Our results indicated that there are no differences in either viability or DCF(+) cells between wild and F1 animals when fresh samples were evaluated. However, when data were analyzed considering two different sperm populations in terms of motility, a significant decrease in viability and DCF(+) cells was reported in low-motile F1 spermatozoa. Cryopreservation did not alter the viability or the presence of DCF(+) cells in sperm samples from wild animals, but significantly decreased the viability in F1 samples. Distribution patterns of H2O2 have been established by confocal microscopy in Solea senegalensis spermatozoa: co-localization of H2O2 with active mitochondria (MitoTracker(+)) and co-localization with nuclear DNA (DAPI). Compared with H2O2 distribution in other marine species such as Scophthalmus maximus, Solea senegalensis spermatozoa showed widespread presence of H2O2 particularly in the nuclei, which could potentially compromise DNA integrity.

Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss).

Zygotic repair of the paternal genome is a key event after fertilization. Spermatozoa accumulate DNA strand breaks during spermatogenesis and can suffer additional damage by different factors, including cryopreservation. Fertilization with DNA-damaged spermatozoa (DDS) is considered to promote implantation failures and abortions, but also long-term effects on the progeny that could be related with a defective repair. Base excision repair (BER) pathway is considered the most active in zygotic DNA repair, but healthy oocytes contain enzymes for all repairing pathways. In this study, the effects of the inhibition of the BER pathway in the zygote were analyzed on the progeny obtained after fertilization with differentially DDS. Massive gene expression (GE; 61 657 unique probes) was analyzed after hatching using microarrays. Trout oocytes are easily fertilized with DDS and the high prolificacy allows live progeny to be obtained even with a high rate of abortions. Nevertheless, the zygotic inhibition of Poly (ADP-ribose) polymerase, upstream of BER pathway, resulted in 810 differentially expressed genes (DEGs) after hatching. DEGs are related with DNA repair, apoptosis, telomere maintenance, or growth and development, revealing a scenario of impaired DNA damage signalization and repair. Downregulation of the apoptotic cascade was noticed, suggesting a selection of embryos tolerant to residual DNA damage during embryo development. Our results reveal changes in the progeny from defective repairing zygotes including higher malformations rate, weight gain, longer telomeres, and lower caspase 3/7 activity, whose long-term consequences should be analyzed in depth.

Accuracy of working length measurement: electronic apex locator versus cone-beam computed tomography.

AIM: To compare the accuracy of working length (WL) determination using the Raypex 6(®) electronic apex locator and cone-beam computed tomography (CBCT). METHODOLOGY: A total of 150 extracted human teeth were decoronated and randomly assigned to five groups (n = 30). WL was measured with the Raypex 6(®) at both the 'constriction' and the 'apex' marks under dry conditions (group 1) or with 2.5% NaOCl, distilled water or Ultracain(®) (groups 2-4). The radiological WL (group 5) was calculated from bucco-lingual and mesio-distal CBCT sections. Differences between electronic, CBCT measurements and actual length (AL) were calculated. Positive and negative values, respectively, indicate measurements falling short or long of AL. Two-way anova and the Bonferroni and Welch tests were used to compare mean differences amongst groups. The chi-squared and Fisher's exact tests were used to compare percentages of precise, ±0.5 and ±1.0 mm of the AL measurements amongst the experimental groups. Statistical analysis was performed at α = 0.05. RESULTS: Mean differences with respect to AL ranged from 0.26 to -0.36 mm and from 0.05 to 0.18 mm, respectively, for the electronic measurements at the 'constriction' mark and 'apex' mark. CBCT measurements were an average of 0.59 mm shorter than AL. Percentages of electronic measurements falling within ±0.5 mm of the corresponding AL referred to the 'apex' mark were greater than at the 'constriction' mark, but the differences were only significant in group 4 (with Ultracain(®) ). Percentages of CBCT measurements falling within ±0.5 mm of AL (46.7%) were significantly lower than electronic measurements, regardless of the condition of the root canal. In 30-38.5% of the measurements taken at the 'apex' mark and in 3.4-13.3% of those at the 'constriction' mark, the file tip extended beyond the foramen. CONCLUSIONS: Electronic measurements were more reliable than CBCT scans for WL determination. The Raypex 6(®) was more accurate in locating the major foramen than the apical constriction under the experimental set-up.

Effect of cryopreservation on human sperm messenger RNAs crucial for fertilization and early embryo development.

During recent years, several studies have pointed out the importance of key paternal transcripts in early embryo development. Sperm cryopreservation is commonly applied in assisted reproductive technologies (ARTs) and it is important to know if it produces any relevant effect at this level. In this study, using normozoospermic donors, we present a candidate transcript approach in which we quantify the presence of sperm mRNAs considered as markers for male fertility and pregnancy success. Analyses were done using quantitative PCR. Our results demonstrated that the used cryopreservation protocol, which is routinely employed in clinical practice, alter transcripts considered as spermatozoa quality markers and markers for pregnancy success. Most of the studied transcripts considered as male quality markers (PRM1, PRM2, and PEG1/MEST) and one of studied mRNAs reported as markers of pregnancy success (ADD1) were reduced after cryopreservation. In order to check if vitrification protocols could reduce this alteration, another assay was carried out analyzing those transcripts with higher differences in the first study (PRM1 and PRM2). The results showed the same tendency. Although maternal mRNAs can compensate these deficiencies, these results could make advisable the optimization of freezing/thawing procedures.

Cryoprotective effects of antifreeze proteins delivered into zebrafish embryos.

Fish embryo cryopreservation, which is useful in aquaculture or biodiversity conservation, is still far from being achieved. Structural barriers reduce the entrance of cryoprotectants into embryo compartments. Previous studies demonstrated a better ability for freezing in Arctic species which naturally express antifreeze proteins (AFPs). In this study, AFPs were delivered in early zebrafish embryos by incubation in media containing protein. Their cryoprotective effects were then analyzed. Chilling sensitivity was evaluated at 4 degrees C and -10 degrees C. Survival rates significantly increased in embryos incorporating AFPI and kept at -10 degrees C. To analyze their effects on cryopreservation, 5-somite embryos were vitrified. Incorporation of AFPI reduced the percentage of embryos that collapsed at thawing (14.2% of AFPI-treated embryos and 48.9% of controls). Cellular damage caused by vitrification was assessed after thawing by cell dissociation and further analysis of cell survival in culture (SYBR-14/IP labeling). The percentage of viable cells at thawing ranged from 25 to 50%, considered incompatible with embryo development. Cells recovered from frozen-control embryos did not survive in culture. However, the incorporation of AFPs allowed survival similar to that of cells recovered from non-frozen embryos. Blastomere cryopreservation trials incorporating AFPI in the extender also demonstrated a significant increase in viability after freezing. Our findings demonstrated that delivery of AFPs into zebrafish embryos by incubation in media containing protein at early stages is a simple and harmless method that increases cryoprotection of the cellular compartment. This beneficial effect is also noticed in blastomeres, encouraging their use in further protocols for embryo cryopreservation.

Estimation of distribution algorithms as logistic regression regularizers of microarray classifiers.

OBJECTIVES: The "large k (genes), small N (samples)" phenomenon complicates the problem of microarray classification with logistic regression. The indeterminacy of the maximum likelihood solutions, multicollinearity of predictor variables and data over-fitting cause unstable parameter estimates. Moreover, computational problems arise due to the large number of predictor (genes) variables. Regularized logistic regression excels as a solution. However, the difficulties found here involve an objective function hard to be optimized from a mathematical viewpoint and a careful required tuning of the regularization parameters. METHODS: Those difficulties are tackled by introducing a new way of regularizing the logistic regression. Estimation of distribution algorithms (EDAs), a kind of evolutionary algorithms, emerge as natural regularizers. Obtaining the regularized estimates of the logistic classifier amounts to maximizing the likelihood function via our EDA, without having to be penalized. Likelihood penalties add a number of difficulties to the resulting optimization problems, which vanish in our case. Simulation of new estimates during the evolutionary process of EDAs is performed in such a way that guarantees their shrinkage while maintaining their probabilistic dependence relationships learnt. The EDA process is embedded in an adapted recursive feature elimination procedure, thereby providing the genes that are best markers for the classification. RESULTS: The consistency with the literature and excellent classification performance achieved with our algorithm are illustrated on four microarray data sets: Breast , Colon , Leukemia and Prostate . Details on the last two data sets are available as supplementary material. CONCLUSIONS: We have introduced a novel EDA-based logistic regression regularizer. It implicitly shrinks the coefficients during EDA evolution process while optimizing the usual likelihood function. The approach is combined with a gene subset selection procedure and automatically tunes the required parameters. Empirical results on microarray data sets provide sparse models with confirmed genes and performing better in classification than other competing regularized methods.

Incorporation of antifreeze proteins into zebrafish embryos by a non-invasive method.

The cryopreservation of fish embryos is a challenge because of their structure, with multiple compartments and permeability barriers, and their high chilling sensitivity. Vitrification at advanced developmental stages is considered to be the more promising option. Nevertheless, all reported attempts have failed. Previous studies demonstrated a better ability for freezing in species that naturally express antifreeze proteins (AFPs). These proteins have been delivered into other fish embryos using time-consuming techniques like microinjection. In the present study, the introduction of FITC labelled AFPs was assayed in zebrafish embryos at early developmental stages (from 2-cell to high blastula stage), before the formation of the yolk syncytial layer, by an easy and non-invasive method and evaluated by fluorescence and confocal microscopy. Incubation with AFPs at 128-cell or high blastula stage provides incorporation of the protein in 50-90% of embryos without affecting hatching. Incubation in media containing protein is a simple, harmless and effective method which makes it possible to treat several embryos at the same time. AFPs remain located in derivatives from marginal blastomeres: the yolk syncytial layer, the most cryosensitive and impermeable barrier, and different digestive organs. Our findings demonstrate that delivery of AFP type I and AFP type III into zebrafish embryos by incubation in media containing protein is a simple and harmless method that may improve cryoprotection of the cellular compartment.

The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures.

To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 degrees C (1h) and -10 degrees C (15min) when embryos reached 5-somite stage. Embryos were subjected to -10 degrees C chilling in a 3M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 degrees C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes.

Probiotic administration improves sperm quality in asthenozoospermic human donors.

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under similar diet conditions participated in the study. The quality of individual sperm samples was evaluated before (previous to ingestion), during (after 3 and 6 weeks of ingestion) and after probiotic administration (3 and 6 weeks after finishing the treatment). Sperm motility was evaluated by computer-assisted sperm analysis system, DNA fragmentation by sperm chromatin structure assay, cell viability by flow cytometry and measurement of intracellular H2O2 (reactive oxygen species; ROS) by flow cytometry using dichloro-dihydrofluorescein diacetate. Sperm motility was drastically improved after the treatment (approximately 6 fold change), DNA fragmentation was statistically reduced after probiotic administration from (approximately 1.2 fold change) and intracellular H2O2 level was decreased (approximately 3.5 fold change). Cell viability was not affected by the treatment. The significant improvement in sperm motility and the decrease in DNA fragmentation reported in this study provide preliminary evidence that probiotics could be administrated to improve motility and decrease DNA fragmentation and ROS levels in asthenozoospermic human males.

[The meaning of PSA progression after radical prostatectomy. Preliminary results]. / Significado de la progresión de PSA tras prostatectomía radical. Resultados preliminares.

OBJECTIVES: To report our findings regarding to the natural history of prostate cancer (PCa) that shows recurrence after radical prostatectomy (RP), in terms of time to development of metastatic disease and death from PCa. To identify independent predictors of PSA recurrence. MATERIAL AND METHODS: Retrospective analysis of 227 patients with clinically localized PCa who underwent RP. The event PSA recurrence was defined as the presence of a postoperative PSA level of 0,2 ng/ml or higher at least 3 months after surgery. Hence, cases with shorter follow-up time were excluded from analysis. No adjuvant therapy (radiotherapy or hormonal therapy) was performed in the included population. Recurrence free survival was calculated during the follow-up period (Kaplan-Meier analysis). Uni and multivariate study was performed in order to assess the ability of factors as preoperative PSA level, Gleason score in surgical specimen, capsular penetration, positive surgical margins (excluding urethral), extracapsular extension, positive pelvic lymph nodes, and seminal vesicle invasion, to predict PSA recurrence. Finally, we selected the group of patients with PSA recurrence and calculated the probability of being free from distant metastatic disease during the follow-up period. Also, function of disease-specific survival was calculated. RESULTS: A total of 208 records were finally included in the study. Median age was 61 years. A total of 47 (22.6%) presented with extracapsular extension. Median follow-up time was 35.8 months, and 49 (23.6%) developed PSA recurrence. Recurrence free survival was 79.9% and 67.4% at 2 and 5 years, respectively. Only three factors were identified with the aid of multivariate analysis as independent predictors of recurrence: preoperative PSA >= 10 ng/ml (hazard ratio--HR--3.03), Gleason score in surgical specimen 8 or higher (HR 3.42), and the finding of capsular penetration (HR 2.17). When only patients with PSA recurrence were considered, 16.3% developed distant metastasis. Probabilities of being free from distant disease after PSA recurrence were 97.7% and 86.9% at 2 and 5 years respectively (actuarial median time 110.8 months). Only 2 patients died from PCa, therefore disease-specific mortality analysis was not performed. CONCLUSIONS: Although an important proportion of patients present with PSA recurrence after RP in our setting, the prognosis in term of development of metastatic disease is acceptable in the short-medium term. Anyway, further analysis will be needed to ascertain the evolution of these patients in the long term.

Selection of nonapoptotic sperm by magnetic-activated cell sorting in Senegalese sole (Solea senegalensis).

Senegalese sole (Solea senegalensis) is a promising species in aquaculture. However, owing to decreased sperm quality in F1 generations and the absence of courtship in those individuals born in captivity, artificial fertilization is being used to generate new progenies. The objective of this study was to implement a sperm selection method for nonapoptotic sperm subpopulation recovery before sperm cryopreservation. In particular, magnetic-activated cell sorting is used to eliminate apoptotic spermatozoa. This study represents the proof-of-concept for magnetic-activated cell sorting applicability in teleost species relevant in aquaculture. Apoptotic cell population was studied by flow cytometry using YO-PRO-1 and a caspase detection kit. Also, reactive oxygen species were measured in sperm samples. Our data demonstrated that caspase detection is more specific than YO-PRO-1 in the identification of apoptotic cells in S senegalensis seminal samples. The results showed that the percentage of apoptotic cells (caspase positive) was significantly higher (P = 0.04) in seminal samples from F1 than that from wild individuals. Magnetic-activated cell sorting removed a significant number of apoptotic cells from the samples (54% and 75% in wild and F1 individuals, respectively), decreasing the level of cells positive for reactive oxygen species (P = 0.17). In conclusion, this technique reduces the percentage of nonfunctional spermatozoa in a seminal sample before cryopreservation. This novel technique can be applied directly in the aquaculture industry.

Current concepts on microscopic colitis: evidence-based statements and recommendations of the Spanish Microscopic Colitis Group.

BACKGROUND: Microscopic colitis (MC) is an underdiagnosed inflammatory bowel disease. AIM: To develop an evidence-based clinical practice guide on MC current concepts. METHODS: Literature search was done on the Cochrane Library, EMBASE and MEDLINE electronic databases, which were consulted covering the period up until March 2015. Work groups were selected for each of the reviewed topics, with the purpose of drafting the initial statements and recommendations. They subsequently underwent a voting process based on the Delphi method. Each statement/recommendation was accompanied by the result of the vote the level of evidence, and discussion of the corresponding evidence. The grade of recommendation (GR) using the GRADE approach was established for diagnosis and treatment recommendations. RESULTS: Some key statements and recommendations are: advancing age increases the risk of developing MC, mainly in females. The symptoms of MC and IBS-D may be similar. If MC is suspected, colonoscopy taking biopsies is mandatory. Treatment with oral budesonide is recommended to induce clinical remission in patients with MC. Oral mesalazine is not recommended in patients with collagenous colitis for the induction of clinical remission. The use of anti-TNF-alpha drugs (infliximab, adalimumab) is recommended for the induction of remission in severe cases of MC that fail to respond to corticosteroids or immunomodulators, as an alternative to colectomy. CONCLUSIONS: This is the first consensus paper on MC based on GRADE methodology. This initiative may help physicians involved in care of these patients in taking decisions based on evidence.

Vitrification assays with embryos from a cold tolerant sub-arctic fish species.

Pseudopleuronectes americanus is a Northern teleost species that produces antifreeze proteins (AFPs) to protect them from freezing during the winter. These AFPs bind to ice crystals to inhibit their growth, and they also protect cell membranes at low temperatures. In this study, vitrification trials were done with fish embryos at three different developmental stages, using two different protocols for incorporating the vitrifying solutions. Toxicity of the cryoprotectants and permeability to dimethyl sulfoxide were analyzed. Embryos were vitrified in 0.5 ml straws by direct immersion in liquid nitrogen, and their morphology and development analyzed following thaw. The embryos responded well to vitrification as evidenced by the high percentage that exhibited good morphology following thaw. Although none of the embryos hatched, a small percentage (0.92%) of them showed active movements within the chorion and continued to develop for a number of days following thaw. This is the first record of post-thaw development of vitrified fish embryos.

[Effect of polyphenols from the Mediterranean diet on proliferation and mediators of in vitro invasiveness of the MB-49 murine bladder cancer cell line]. / Efecto de polifenoles de la dieta mediterránea sobre la proliferación y mediadores de la invasividad "in vitro" de la linea de cancer vesical murino MB-49.

OBJECTIVE: To evaluate the effect of different polyphenols on the proliferation and invasive capacity of MB-49 murine bladder tumor cell lines and to identify the mediators involved in this process. MATERIALS AND METHODS: MB-49 murine bladder cancer cells were cultured in media supplemented with resveratrol, rutin, morin, quercetin, gallic acid and tannic acid (all of them are polyphenols usually present in Mediterranean diet) for periods of 24, 48 and 72 hours to quantify the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in the culture medium, as well as of metalloproteinase-9 (MMP-9) and cell proliferation. RESULTS: All the polyphenols studied significantly inhibited proliferation of MB-49 cells, varying according to the time periods and doses used. The cells in the media supplemented with the nutrients to study did not show inhibition of mRNA expression of urokinase-type plasminogen activator (uPA) or its high affinity receptor (uPAR). It was even slightly increased in certain cases. However, mRNA expression of metalloproteinase-9 was strongly inhibited. CONCLUSIONS: The polyphenols present in our usual diet exert an effect on the proliferation and mediators of bladder tumor invasiveness in MB-49 cells.

La cirugía en enfermedad aortoiliaca. ¿tiene un lugar en la era endovascular? / Surgery in aortoiliac disease. do you have a place in the endovascular era?

INTRODUCCIÓN: La enfermedad aorto iliaca con clasificación TASC II tipo C deja abierta las alternativas para manejo Endovascular vs cirugía abierta. El seleccionar la mejor estrategia ayuda a disminuir la morbimortalidad. OBJETIVOS: Presentar nuestra experiencia en el tratamiento de pacientes con enfermedad aorto iliaca. MÉTODOS: De julio 2017 a julio del 2018 se realizó una revisión de expediente en busca de enfermedad aorto iliaca TASC II tipo C encontrando 7 pacientes. RESULTADOS: La edad promedio 61.7 años, con un rango promedio de 46 a 70 años, 71% del sexo femenino y 29% masculino, antecedentes de diabetes mellitus 3 (43%) y 4 (57%) con hipertensión arterial, tabaquismo en 6 pacientes que corresponde al 86%, en todos los pacientes estaba presente claudicación intermitente, en 5 pacientes (72%) lesiones necróticas en las extremidades inferiores. Obteniendo una mejoría en relación a su toma inicial de 0.18 MPD y 0.14 para MPI, ningún paciente requirió reintervención no hubo casos de oclusión del injerto. Solo se observaron dos complicaciones menores con un éxito técnicos a los 6 meses de seguimiento post quirúrgico. CONCLUSIONES: La cirugía abierta en enfermedad aorto iliaca TASC II lesiones tipo C puede ser una estrategia terapéutica viable, definitiva y menos costosa

BACKGROUND: Iliac aorto disease with TASC II type C classification leaves open alternatives for endovascular vs open surgery management. Selecting the best strategy helps reduce morbidity and mortality. OBJECTIVES: Present our experience in the treatment of patients with iliac aorto disease. METHOD: From July 2017 to July 2018, we treated 7 patients with TASC II Type A Aortoiliary disease. On admission, we performed a clinical history, assessment of the degree of claudication, palpation of pulses of the lower extremity, description of the type of necrotic lesions in the foot, ankle / arm index shot and Angio 3D tomography study. Patients with aorto iliac type A, B and D lesions were excluded. Open surgery was performed with bifurcated aorto-iliac or aorto-bi-femoral aortic graft, the follow-up was given by the external consultation, ankle / arm index monitoring, improvement of the claudication and limitation of the area of necrosis. RESULTS: The average age was 61.7 years, with an average range of 46 to 70 years, 71% of the female sex and 29% male, a history of diabetes mellitus 3 (43%) and 4 (57%) with hypertension, smoking in 6 patients corres-ponding to 86%, intermittent claudication was present in all patients, in 5 patients (72%) necrotic lesions in the lower extremities. The average ankle /arm index before and after surgery for right leg 0.62 to 0.8 respectively and for left leg 0.58 to 0.72. Obtaining an improvement in relation to its initial intake of 0.18 MPD and 0.14 for MPI, no patient required re-intervention and there were no cases of graft occlusion. Only two minor complications were observed with a technical success at 3 months post-surgical follow-up. CONCLUSIONS: Open surgery in aorto iliaca disease TASC II type C lesions can be a therapeutic strategy when selecting the right patient. Hybrid treatments; open surgery and endovascular are allowing better results so it is important not to abandon this technique in our patients

Azacitidine frontline therapy for unfit acute myeloid leukemia patients: clinical use and outcome prediction.

Hypomethylating agents are able to prolong the overall survival of some patients diagnosed with acute myeloid leukemia. The aim of this study was to evaluate the clinical use of azacitidine as front-line therapy in unfit acute myeloid leukemia (AML) patients and to develop a clinical prediction model to identify which patients may benefit more from the drug. One hundred and ten untreated unfit AML patients received front-line azacitidine therapy in Spain, and response and survival were evaluated in them following European LeukemiaNet (ELN) guidelines. A clinical prediction rule was obtained from this population that was validated and refined in 261 patients treated in France, Austria and Italy. ELN response was achieved in 21.0% of the 371 patients (CI95% 17.0-25.5) and did not depend on bone marrow blast cell percentage. Median overall survival was 9.6 months (CI95% 8.5-10.8) and 40.6% of the patients were alive at 1 year (CI95% 35.5-45.7). European ALMA score (E-ALMA), based on performance status, white blood cell counts at azacitidine onset and cytogenetics, discriminated three risk groups with different survival and response rates. Azacitidine seems a reasonable therapeutic option for most unfit AML patients, i.e. those displaying a favorable or intermediate E-ALMA score.

Dimethyl sulfoxide influx in turbot embryos exposed to a vitrification protocol.

The particular characteristics of fish embryos require the development of specific methods for cryopreservation. One of the main obstacles is related to the presence of membranes and compartments with different water and cryoprotectant permeability. To assess dimethyl sulfoxide (Me2SO4) permeability, we exposed turbot embryos (Scophthalmus maximus) at F stage (tail bud) to the cryoprotectant solutions used in a vitrification protocol and then evaluated the Me2SO4 content inside the embryo using high-performance liquid chromatography (HPLC). The Me2SO4 influx was analyzed in normal embryos and in embryos treated with pronase (2mg/ml) in order to increase chorion permeability. The evaluation was made after each step of cryoprotectant incorporation and removal. Three embryo compartments were distinguished: the perivitelline space (PVS), the yolk sac (YS) and the cellular compartment (CC), and the relative volumes of each, estimated using stereoscopic microscopy imaging, were 11.37, 81.23 and 7.40%, respectively. The Me2SO4 concentration inside the embryos was calculated based on their entrance into one, two or three compartments. Results suggest high entrance of Me2SO4 into the PVS and a low concentration of this cryoprotectant inside the other compartments. Pronase did not significantly increase Me2SO4 influx, but facilitated its elimination during the washing steps.

Effect of a vitrification protocol on the lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities and the hatching rates of Zebrafish (Danio rerio) and Turbot (Scophthalmus maximus) embryos.

Vitrification, is the most promising option for the cryopreservation of fish embryos but requires high concentrations of potentially toxic cryoprotectants. In this study, embryos from Turbot and Zebrafish, each in two developmental stages, were submitted to a four stepwise cryoprotectant incorporation protocol. After incubation in the vitrificant solution (5M dimethyl sulfoxide, 2M methanol, 1M ethylen-glycol and 10% sucrose) embryos were loaded in straws and plunged into liquid nitrogen. The activity of two cytoplasmic enzymes, LDH and G6PDH, and the hatching rates were analyzed in control embryos, those subjected to the cryoprotectant solutions and in frozen/thawed embryos. Results showed that the cryoprotectants incorporation protocol did not have important effects on the analyzed enzymatic activities, which remained at similar levels to that in control embryos but significantly reduced the hatching rates. Turbot was less sensitive than Zebrafish to the toxic effect of the cryoprotectants, achieving hatching rates of 74.8% in comparison with fresh control embryos at G stage, whereas in Zebrafish only 17.7% of hatching was reported with five somites-treated embryos. In Turbot, G stage was more resistant to the cryoprotectants and thus more convenient for further vitrification studies. After vitrification no survival was recorded and enzymatic activities dropped significantly, particularly in Zebrafish, indicating cell damage and loss of cytoplasmic enzymes. Nevertheless, total cell lysis was not produced, and once again Turbot was more resistant to the effect of vitrification, particularly at the later stage. In that stage, Turbot embryos showed around 50% of G6PDH activity after vitrification, in comparison with the control, indicating the preservation of some cellular activity after freezing-thawing, despite the loss of developmental ability.

[Epidemiologic analysis of clear cell and chromophilic carcinoma of the kidney]. / Análisis epidemiológico del carcinoma renal de células claras y cromofílico.

OBJECTIVE: Finding differences in the characteristics of the two most frequent variants of renal cancers: the clear-cell renal carcinoma, and the chromophilic one. MATERIAL AND METHODS: Retrospective analysis of epidemiological characteristics of patients diagnosed of renal carcinoma in our hospital between 1991 and 2001. Statistical differences were searched between patients' characteristics (age, sex, smoking habitus) and tumors' characteristics (size, focality, stage, side and grade). RESULTS: Sixty six renal tumors were diagnosed, of which 41 (62.1%) were clear-cell tumors and 9 (13%) were chromophilic. We only found statistical differences between both subpopulations' tumor size (p < 0.05), being greater for clear-cell tumors (7 +/- 3.92 cm) than for chromophilic ones (4.89 +/- 1.96 cm). We could also appreciate a bias towards a less advanced stage of the chromophilic type, although not statistically significant. CONCLUSIONS: Clear-cell renal carcinoma and chromophilic renal carcinoma are the two more frequent variants of renal tumors. The chromophilic type is smaller and is usually found in a less advanced stage, although this bias could not be demonstrated in our series.